Compositions and methods for conjugating activatable antibodies

ABSTRACT

The invention relates generally to compositions and methods for conjugating antibodies and activatable antibodies, and methods of partially reducing antibodies and/or activatable antibodies prior to conjugation, e.g., thiol-based conjugation, with an agent, e.g., a therapeutic and/or diagnostic agent.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/296,207, filed Jun. 4, 2014, which claims the benefit of U.S.Provisional Application No. 61/830,913, filed Jun. 4, 2013 and U.S.Provisional Application No. 61/919,935, filed Dec. 23, 2013. Thecontents of each of which are hereby incorporated by reference in theirentirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “CYTM023D01USSeqList.txt”, which wascreated on Nov. 22, 2016 and is 160 KB in size, are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods forconjugating antibodies and activatable antibodies, and methods ofpartially reducing antibodies and/or activatable antibodies prior toconjugation, e.g., thiol-based conjugation, with an agent, e.g., atherapeutic and/or diagnostic agent.

BACKGROUND OF THE INVENTION

Antibody-based therapies have proven effective treatments for somediseases but in some cases, toxicities due to broad target expressionhave limited their therapeutic effectiveness. In addition,antibody-based therapeutics have exhibited other limitations such asrapid clearance from the circulation following administration.Conjugating agents to antibodies has been used to further advance theuse of antibody-based therapies. Molecules such as toxins, radionuclidesand drugs including anti-cancer drugs have been conjugated to certainantibodies to generate immunotoxins, radioimmunoconjugates, and/orantibody-drug conjugates (ADCs).

In the realm of small molecule therapeutics, strategies have beendeveloped to provide prodrugs of an active chemical entity. Suchprodrugs are administered in a relatively inactive (or significantlyless active) form. Once administered, the prodrug is metabolized in vivointo the active compound. Such prodrug strategies can provide forincreased selectivity of the drug for its intended target and for areduction of adverse effects.

Accordingly, there is a continued need in the field of antibody-basedtherapeutics for antibodies that mimic the desirable characteristics ofthe small molecule prodrug, as well as a need for improved methods ofconjugating agents to these antibodies without negatively impactingtheir ability to mimic the desirable characteristics of the smallmolecule prodrug.

SUMMARY OF THE INVENTION

The present invention provides conjugates that include an activatableantibody and methods of making these activatable antibody conjugates.Also provided are activatable antibodies having points of conjugationfor receiving a drug or label. The conjugates can be usedtherapeutically, diagnostically (e.g., in vitro or in vivo), for in vivoimaging, and for any of a variety of other therapeutic, diagnosticand/or prophylactic uses.

Generally, the compositions and methods provided herein include anactivatable antibody that includes an antibody or antibody fragment (AB)that specifically binds a target, where the AB is coupled to a maskingmoiety (MM) that decreases the ability of the AB to bind its target. Insome embodiments, the activatable antibody further includes a cleavablemoiety (CM) that is a substrate for a protease. The compositions andmethods provided herein enable the attachment of one or more agents toone or more cysteine residues in the AB without compromising theactivity (e.g., the masking, activating or binding activity) of theactivatable antibody. In some embodiments, the compositions and methodsprovided herein enable the attachment of one or more agents to one ormore cysteine residues in the AB without reducing or otherwisedisturbing one or more disulfide bonds within the MM. The compositionsand methods provided herein produce an activatable antibody that isconjugated to one or more agents, e.g., any of a variety of therapeutic,diagnostic and/or prophylactic agents, and, in some embodiments, withoutany of the agent(s) being conjugated to the MM of the activatableantibody. The compositions and methods provided herein produceconjugated activatable antibodies in which the MM retains the ability toeffectively and efficiently mask the AB of the activatable antibody inan uncleaved state. The compositions and methods provided herein produceconjugated activatable antibodies in which the activatable antibody isstill activated, i.e., cleaved, in the presence of a protease that cancleave the CM.

The activatable antibodies have at least one point of conjugation for anagent, but in the methods and compositions provided herein less than allpossible points of conjugation are available for conjugation to anagent. In some embodiments, the one or more points of conjugation aresulfur atoms involved in disulfide bonds. In some embodiments, the oneor more points of conjugation are sulfur atoms involved in interchaindisulfide bonds. In some embodiments, the one or more points ofconjugation are sulfur atoms involved in interchain sulfide bonds, butnot sulfur atoms involved in intrachain disulfide bonds. In someembodiments, the one or more points of conjugation are sulfur atoms ofcysteine or other amino acid residues containing a sulfur atom. Suchresidues may occur naturally in the antibody structure or may beincorporated into the antibody by site-directed mutagenesis, chemicalconversion, or mis-incorporation of non-natural amino acids.

Also provided are methods of preparing a conjugate of an activatableantibody having one or more interchain disulfide bonds in the AB and oneor more intrachain disulfide bonds in the MM, and a drug reactive withfree thiols is provided. The method generally includes partiallyreducing interchain disulfide bonds in the activatable antibody with areducing agent, such as, for example, TCEP; and conjugating the drugreactive with free thiols to the partially reduced activatable antibody.As used herein, the term partial reduction refers to situations where anactivatable antibody is contacted with a reducing agent and less thanall disulfide bonds, e.g., less than all possible sites of conjugationare reduced. In some embodiments, less than 99%, 98%/0, 97%, 96%, 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%/0, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10% or less than 5% of all possible sites of conjugation arereduced.

In some embodiments, a method of reducing and conjugating an agent,e.g., a drug, to an activatable antibody resulting in selectivity in theplacement of the agent is provided. The method generally includespartially reducing the activatable antibody with a reducing agent suchthat any conjugation sites in the masking moiety or other non-AB portionof the activatable antibody are not reduced, and conjugating the agentto interchain thiols in the AB. The conjugation site(s) are selected soas to allow desired placement of an agent to allow conjugation to occurat a desired site. The reducing agent is, for example, TCEP. Thereduction reaction conditions such as, for example, the ratio ofreducing agent to activatable antibody, the length of incubation, thetemperature during the incubation, the pH of the reducing reactionsolution, etc., are determined by identifying the conditions thatproduce a conjugated activatable antibody in which the MM retains theability to effectively and efficiently mask the AB of the activatableantibody in an uncleaved state. The ratio of reduction agent toactivatable antibody will vary depending on the activatable antibody. Insome embodiments, the ratio of reducing agent to activatable antibodywill be in a range from about 20:1 to 1:1, from about 10:1 to 1:1, fromabout 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, fromabout 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, fromabout 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, fromabout 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5,from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In someembodiments, the ratio is in a range of from about 5:1 to 1:1. In someembodiments, the ratio is in a range of from about 5:1 to 1.5:1. In someembodiments, the ratio is in a range of from about 4:1 to 1:1. In someembodiments, the ratio is in a range from about 4:1 to 1.5:1. In someembodiments, the ratio is in a range from about 8:1 to about 1:1. Insome embodiments, the ratio is in a range of from about 2.5:1 to 1:1.

In some embodiments, a method of reducing interchain disulfide bonds inthe AB of an activatable antibody and conjugating an agent, e.g., athiol-containing agent such as a drug, to the resulting interchainthiols to selectively locate agent(s) on the AB is provided. The methodgenerally includes partially reducing the AB with a reducing agent toform at least two interchain thiols without forming all possibleinterchain thiols in the activatable antibody; and conjugating the agentto the interchain thiols of the partially reduced AB. For example, theAB of the activatable antibody is partially reduced for about 1 hour atabout 37° C. at a desired ratio of reducing agent:activatable antibody.In some embodiments, the ratio of reducing agent to activatable antibodywill be in a range from about 20:1 to 1:1, from about 10:1 to 1:1, fromabout 9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, fromabout 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, fromabout 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, fromabout 10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5,from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In someembodiments, the ratio is in a range of from about 5:1 to 1:1. In someembodiments, the ratio is in a range of from about 5:1 to 1.5:1. In someembodiments, the ratio is in a range of from about 4:1 to 1:1. In someembodiments, the ratio is in a range from about 4:1 to 1.5:1. In someembodiments, the ratio is in a range from about 8:1 to about 1:1. Insome embodiments, the ratio is in a range of from about 2.5:1 to 1:1.

The thiol-containing reagent can be, for example, cysteine or N-acetylcysteine. The reducing agent can be, for example, TCEP. In someembodiments, the reduced activatable antibody can be purified prior toconjugation, using for example, column chromatography, dialysis, ordiafiltration. In some embodiments, the reduced antibody is not purifiedafter partial reduction and prior to conjugation.

In some embodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof (AB) that specifically binds a target,wherein the AB is coupled to a masking moiety (MM), such that couplingof the MM to the AB decreases the ability of the antibody orantigen-binding fragment thereof to bind the target. In someembodiments, the MM is coupled to the AB via a cleavable moiety (CM)that includes a substrate for a protease, for example, a protease thatis co-localized with the target at a treatment site in a subject. Theactivatable antibodies provided herein are stable in circulation,activated at intended sites of therapy and/or diagnosis but not innormal, e.g., healthy, tissue, and, when activated, exhibit binding tothe target that is at least comparable to the corresponding, unmodifiedantibody.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof (AB) that specifically binds thetarget. In some embodiments, the antibody or immunologically activefragment thereof that binds the target is a monoclonal antibody, domainantibody, single chain, Fab fragment, a F(ab′)₂ fragment, a scFv, ascAb, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, such an antibody orimmunologically active fragment thereof that binds the target is amouse, chimeric, humanized or fully human monoclonal antibody. In someembodiments, the antigen binding fragment thereof is a Fab fragment, aF(ab′)₂ fragment, a scFv, or a scAb.

In some embodiments, the antibody or an antigen binding fragment thereof(AB) specifically binds to a target selected from those shown Table 1.In some embodiments, the AB specifically binds to Epidermal GrowthFactor Receptor (EGFR). In some embodiments, the AB specifically bindsto Jagged 1 and/or Jagged 2. In some embodiments, the AB specificallybinds to interleukin 6 receptor (IL-6R).

In some embodiments, the antibody or an antigen binding fragment thereof(AB) is or is derived from an antibody selected from those shown inTable 2.

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to the target.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is no more than the equilibrium dissociationconstant of the AB to the target.

In some embodiments, the MM does not interfere or compete with the AB ofthe activatable antibody in a cleaved state for binding to the target.

In some embodiments, the MM is a polypeptide of about 2 to 40 aminoacids in length, for example, no more than 40 amino acids long.

In some embodiments, the MM polypeptide sequence is different from thatof the target, and the MM polypeptide sequence is no more than 500identical to any natural binding partner of the AB.

In some embodiments, the MM polypeptide sequence is different from thatof the target, and the MM polypeptide sequence is no more than 25%identical to any natural binding partner of the AB. In some embodiments,the MM polypeptide sequence is different from that of the target, andthe MM polypeptide sequence is no more than 10% identical to any naturalbinding partner of the AB.

In some embodiments, the coupling of the MM to the AB decreases theability of the AB to bind the target such that the dissociation constant(K_(d)) of the AB when coupled to the MM towards the target is at least20 times greater than the K_(d) of the AB when not coupled to the MMtowards the target. In some embodiments, the coupling of the MM to theAB decreases the ability of the AB to bind the target such that thedissociation constant (K_(d)) of the AB when coupled to the MM towardsthe target is at least 40 times greater than the K_(d) of the AB whennot coupled to the MM towards the target. In some embodiments, thecoupling of the MM to the AB decreases the ability of the AB to bind thetarget such that the dissociation constant (K_(d)) of the AB whencoupled to the MM towards the target is at least 50 times greater thanthe K_(d) of the AB when not coupled to the MM towards the target. Insome embodiments, the coupling of the MM to the AB decreases the abilityof the AB to bind the target such that the K_(d) of the AB when coupledto the MM towards the target is at least 100 times greater than theK_(d) of the AB when not coupled to the MM towards the target. In someembodiments, the coupling of the MM to the AB decreases the ability ofthe AB to bind the target such that the K_(d) of the AB when coupled tothe MM towards the target is at least 1000 times greater than the K_(d)of the AB when not coupled to the MM towards the target. In someembodiments, the coupling of the MM to the AB decreases the ability ofthe AB to bind the target such that the K_(d) of the AB when coupled tothe MM towards the target is at least 10,000 times greater than theK_(d) of the AB when not coupled to the MM towards the target.

In some embodiments, in the presence of the target, the MM decreases theability of the AB to bind the target by at least 90% when the CM isuncleaved, as compared to when the CM is cleaved when assayed in vitrousing a target displacement assay such as, for example, the assaydescribed in PCT Publication Nos. WO 2009/025846 and WO 2010/081173.

In some embodiments, the protease is co-localized with the target in atissue, and the protease cleaves the CM in the activatable antibody whenthe activatable antibody is exposed to the protease. In someembodiments, the protease is not active or is significantly less activein tissues that do not significantly express the target. In someembodiments, the protease is not active or is significantly less activein healthy, e.g., non-diseased tissues.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, the CM is a substrate for a protease selected fromthe group consisting of those shown in Table 3. In some embodiments, theCM is a substrate for a protease selected from the group consisting ofuPA (urokinase plasminogen activator), legumain and MT-SP1 (matriptase).In some embodiments, the protease comprises uPA. In some embodiments,the protease comprises legumain. In some embodiments, the proteasecomprises MT-SP1.

In some embodiments, the CM is a substrate for at least two proteases.In some embodiments, each protease is selected from the group consistingof those shown in Table 3. In some embodiments, the CM is a substratefor at least two proteases, and one of the proteases is selected fromthe group consisting of uPA, legumain and MT-SP1 and the other proteaseis selected from the group consisting of those shown in Table 3. In someembodiments, the CM is a substrate for at least two proteases selectedfrom the group consisting of uPA, legumain and MT-SP1.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is decreased such that it occurs with an equilibriumdissociation constant that is at least 20-fold greater than theequilibrium dissociation constant of an unmodified AB binding to thetarget, and whereas the AB of the activatable antibody in the cleavedstate binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is decreased such that it occurs with an equilibriumdissociation constant that is at least 40-fold greater than theequilibrium dissociation constant of an unmodified AB binding to thetarget, and whereas the AB of the activatable antibody in the cleavedstate binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is decreased such that it occurs with an equilibriumdissociation constant that is at least 50-fold greater than theequilibrium dissociation constant of an unmodified AB binding to thetarget, and whereas the AB of the activatable antibody in the cleavedstate binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is decreased such that it occurs with an equilibriumdissociation constant that is at least 100-fold greater than theequilibrium dissociation constant of an unmodified AB binding to thetarget, and whereas the AB of the activatable antibody in the cleavedstate binds the target.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody tothe target is decreased such that it occurs with an equilibriumdissociation constant that is at least 200-fold greater than theequilibrium dissociation constant of an unmodified AB binding to thetarget, and whereas the AB of the activatable antibody in the cleavedstate binds the target.

In some embodiments, the activatable antibody includes a linking peptidebetween the MM and the CM.

In some embodiments, the activatable antibody includes a linking peptidebetween the CM and the AB.

In some embodiments, the activatable antibody includes a first linkingpeptide (LP1) and a second linking peptide (LP2), and the activatableantibody in the uncleaved state has the structural arrangement fromN-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB orAB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need notbe identical to each other.

In some embodiments, each of LP1 and LP2 is a peptide of about 1 to 20amino acids in length.

In some embodiments, at least one of LP1 or LP2 includes an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 21) and (GGGS)_(n) (SEQ ID NO: 22), where n isan integer of at least one. In some embodiments, at least one of LP1 orLP2 includes an amino acid sequence selected from the group consistingof GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG (SEQ ID NO: 25),GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and GSSSG (SEQ ID NO: 28).

In some embodiments, the activatable antibody is exposed to and cleavedby a protease such that, in the activated or cleaved state, theactivated antibody includes a light chain amino acid sequence thatincludes at least a portion of LP2 and/or CM sequence after the proteasehas cleaved the CM.

In some embodiments, the activatable antibody also includes a signalpeptide. In some embodiments, the signal peptide is conjugated to theactivatable antibody via a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody.

In some embodiments, the activatable antibody in an uncleaved statecomprises a spacer that is joined directly to the MM and has thestructural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.In some embodiments, the spacer includes at least the amino acidsequence QGQSGQ (SEQ ID NO: 11).

In some embodiments, the AB of the activatable antibody naturallycontains one or more disulfide bonds. In some embodiments, the AB can beengineered to include one or more disulfide bonds.

In some embodiments, the agent conjugated to the activatable antibody isa therapeutic agent. In some embodiments, the agent conjugated to theactivatable antibody is a diagnostic agent. In some embodiments, theagent conjugated to the activatable antibody is a prophylactic agent.

In some embodiments, the agent is an antineoplastic agent. In someembodiments, the agent is a toxin or fragment thereof. As used herein, afragment of a toxin is a fragment that retains toxic activity. In someembodiments, the agent is an agent selected from the group listed inTable 4. In some embodiments, the agent is a microtubule inhibitor. Insome embodiments, the agent is a dolastatin. In some embodiments, theagent is an auristatin or derivative thereof. In some embodiments, theagent is auristatin E or a derivative thereof. In some embodiments, theagent is monomethyl auristatin E (MMAE). In some embodiments, the agentis monomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or maytansinoid derivative. In some embodiments, the agentis DM1 or DM4. In some embodiments, the agent is a nucleic acid damagingagent. In some embodiments, the agent is a duocarmycin or derivativethereof. In some embodiments, the agent is a calicheamicin or derivativethereof.

In some embodiments, the agent is conjugated to the AB via a linker. Insome embodiments, the linker is a thiol-containing linker. In someembodiments, the linker is a cleavable linker. In some embodiments, thelinker is selected from the group consisting of the linkers shown inTables 5 and 6.

In some embodiments, the activatable antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent. In some embodiments, the detectable moiety is a conjugatabledetection reagent. In some embodiments, the detectable moiety is, forexample, a fluorophore, for example, a fluorescein derivative such asfluorescein isothiocyanate (FITC).

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody is monospecific. In some embodiments, theactivatable antibody and/or conjugated activatable antibody ismultispecific, e.g., by way of non-limiting example, bispecific ortrifunctional. In some embodiments, the activatable antibody and/orconjugated activatable antibody is formulated as part of apro-Bispecific T Cell Engager (BITE) molecule. In some embodiments, theactivatable antibody and/or conjugated activatable antibody isformulated as part of a pro-Chimeric Antigen Receptor (CAR) modified Tcell or other engineered receptor.

In some embodiments, the serum half-life of the activatable antibody islonger than that of the corresponding antibody; e.g., the pK of theactivatable antibody is longer than that of the corresponding antibody.In some embodiments, the serum half-life of the activatable antibody issimilar to that of the corresponding antibody. In some embodiments, theserum half-life of the activatable antibody is at least 15 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 12 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 11 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least10 days when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 9 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 8 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 7 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 days when administered to an organism. In some embodiments, the serumhalf-life of the conjugated activatable antibody is at least 5 days whenadministered to an organism. In some embodiments, the serum half-life ofthe conjugated activatable antibody is at least 4 days when administeredto an organism. In some embodiments, the serum half-life of theconjugated activatable antibody is at least 3 days when administered toan organism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 2 days when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 24 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 20 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 18 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 16 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 14 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 12 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 10 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 8 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 6 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 4 hours when administered to anorganism. In some embodiments, the serum half-life of the conjugatedactivatable antibody is at least 3 hours when administered to anorganism.

The invention provides partially reduced activatable antibodies in whichat least one interchain disulfide bond in the activatable antibody hasbeen reduced with a reducing agent without disturbing any intrachaindisulfide bonds in the activatable antibody, wherein the activatableantibody includes an antibody or an antigen binding fragment thereof(AB) that specifically binds to a target, a masking moiety (MM) thatinhibits the binding of the AB of the activatable antibody in anuncleaved state to the target, and a cleavable moiety (CM) coupled tothe AB, and the CM is a polypeptide that functions as a substrate for aprotease. In some embodiments, one or more intrachain disulfide bond(s)of the activatable antibody is not disturbed by the reducing agent. Insome embodiments, one or more intrachain disulfide bond(s) of the MMwithin the activatable antibody is not disturbed by the reducing agent.In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP.

The invention also provides partially reduced activatable antibodies inwhich at least one interchain disulfide bond in the activatable antibodyhas been reduced with a reducing agent without disturbing or otherwisecompromising the activity and/or efficacy of the activatable antibody,wherein the activatable antibody includes an antibody or an antigenbinding fragment thereof (AB) that specifically binds to a target, amasking moiety (MM) that inhibits the binding of the AB of theactivatable antibody in an uncleaved state to the target, and acleavable moiety (CM) coupled to the AB, and the CM is a polypeptidethat functions as a substrate for a protease. The activity and/orefficacy of the activatable antibody is, by way of nonlimiting example,masking activity, activation of the activatable antibody, and/or bindingactivity of the activated activatable antibody. In some embodiments, oneor more intrachain disulfide bond(s) of the activatable antibody is notdisturbed by the reducing agent. In some embodiments, one or moreintrachain disulfide bond(s) of the MM within the activatable antibodyis not disturbed by the reducing agent. In some embodiments, theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM. In some embodiments, reducing agent is TCEP.

In some embodiments, the partially reduced activatable antibody isconjugated to an agent through at least one interchain thiol. In someembodiments, the agent is selected from the group of agents listed inTable 4. In some embodiments, the agent is a toxin or fragment thereof.In some embodiments, the agent is a microtubule inhibitor. In someembodiments, the agent is a dolastatin. In some embodiments, the agentis an auristatin or derivative thereof. In some embodiments, the agentis auristatin E or a derivative thereof. In some embodiments, the agentis monomethyl auristatin E (MMAE). In some embodiments, the agent ismonomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or a derivative thereof. In some embodiments, the agent isDM1 or DM4. In some embodiments, the agent is a nucleic acid damagingagent. In some embodiments, the agent is a duocarmycin or derivativethereof. In some embodiments, the agent is a calicheamicin or derivativethereof. In some embodiments, the agent is conjugated to the AB via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the agent is a detectable moiety. In some embodiments, thedetectable moiety is a diagnostic agent.

In some embodiments, the target is selected from the group of targetslisted in Table 1. In some embodiments, the AB is or is derived from anantibody selected from the group of antibodies listed in Table 2. Insome embodiments, the antigen binding fragment thereof is selected fromthe group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, ascAb, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, the AB has an equilibriumdissociation constant of about 100 nM or less for binding to the target.In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to the target. In some embodiments, the MM isa polypeptide of no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget, and the MM polypeptide sequence is no more than 50%/o identicalto any natural binding partner of the AB. In some embodiments, the MMdoes not include more than 25% amino acid sequence identity to thetarget. In some embodiments, the MM does not include more than 10% aminoacid sequence identity to the target. In some embodiments, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments, theprotease is co-localized with the target in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the activatableantibody includes a linking peptide between the MM and the CM. In someembodiments, the activatable antibody includes a linking peptide betweenthe CM and the AB. In some embodiments, the activatable antibodyincludes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in an uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, at least one of LP1 or LP2includes an amino acid sequence selected from the group consisting of(GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 21) and (GGGS)_(n)(SEQ IDNO: 22), where n is an integer of at least one. In some embodiments, atleast one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24),GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), andGSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate for anenzyme selected from the group consisting of those shown in Table 3. Insome embodiments, the activatable antibody in an uncleaved stateincludes a spacer, and the spacer is joined directly to the MM and hasthe structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.

The invention also provides partially reduced activatable antibodies inwhich at least one disulfide bond between the activatable antibody and asecond molecule has been reduced with a reducing agent withoutdisturbing any intrachain disulfide bonds in the activatable antibody,wherein the activatable antibody includes an antibody or an antigenbinding fragment thereof (AB) that specifically binds to a target, amasking moiety (MM) that inhibits the binding of the AB of theactivatable antibody in an uncleaved state to the target, and acleavable moiety (CM) coupled to the AB, and the CM is a polypeptidethat functions as a substrate for a protease. In some embodiments, thesecond molecule is cysteine. In some embodiments, the second molecule isglutathione.

The invention also provides partially reduced activatable antibodies inwhich at least one disulfide bond between the activatable antibody and asecond molecule has been reduced with a reducing agent withoutdisturbing or otherwise compromising the activity and/or efficacy of theactivatable antibody, wherein the activatable antibody includes anantibody or an antigen binding fragment thereof (AB) that specificallybinds to a target, a masking moiety (MM) that inhibits the binding ofthe AB of the activatable antibody in an uncleaved state to the target,and a cleavable moiety (CM) coupled to the AB, and the CM is apolypeptide that functions as a substrate for a protease. The activityand/or efficacy of the activatable antibody is, by way of nonlimitingexample, masking activity, activation of the activatable antibody,and/or binding activity of the activated activatable antibody. In someembodiments, the second molecule is cysteine. In some embodiments, thesecond molecule is glutathione.

In some embodiments, the partial reduction method does not disturb oneor more intrachain disulfide bonds of the activatable antibody. In someembodiments, the method does not disturb one or more intrachaindisulfide bonds of the MM within the activatable antibody.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP.

In some embodiments, the partially reduced activatable antibody isconjugated to an agent through at least one thiol. In some embodiments,the agent is selected from the group of agents listed in Table 4. Insome embodiments, the agent is a toxin or fragment thereof. In someembodiments, the agent is a microtubule inhibitor. In some embodiments,the agent is a dolastatin. In some embodiments, the agent is anauristatin or derivative thereof. In some embodiments, the agent isauristatin E or a derivative thereof. In some embodiments, the agent ismonomethyl auristatin E (MMAE). In some embodiments, the agent ismonomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or a derivative thereof. In some embodiments, the agent isDM1 or DM4. In some embodiments, the agent is a nucleic acid damagingagent. In some embodiments, the agent is a duocarmycin or derivativethereof. In some embodiments, the agent is a calicheamicin or derivativethereof. In some embodiments, the agent is conjugated to the AB via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the agent is a detectable moiety. In some embodiments, thedetectable moiety is a diagnostic agent.

In some embodiments, the target is selected from the group of targetslisted in Table 1. In some embodiments, the AB is or is derived from anantibody selected from the group of antibodies listed in Table 2. Insome embodiments, the antigen binding fragment thereof is selected fromthe group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, ascAb, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, the AB has an equilibriumdissociation constant of about 100 nM or less for binding to the target.In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to the target. In some embodiments, the MM isa polypeptide of no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget, and the MM polypeptide sequence is no more than 50% identical toany natural binding partner of the AB. In some embodiments, the MM doesnot include more than 25% amino acid sequence identity to the target. Insome embodiments, the MM does not include more than 10% amino acidsequence identity to the target. In some embodiments, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments, theprotease is co-localized with the target in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the activatableantibody includes a linking peptide between the MM and the CM. In someembodiments, the activatable antibody includes a linking peptide betweenthe CM and the AB. In some embodiments, the activatable antibodyincludes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in an uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, at least one of LP1 or LP2includes an amino acid sequence selected from the group consisting of(GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 21) and (GGGS)_(n)(SEQ IDNO: 22), where n is an integer of at least one. In some embodiments, atleast one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24),GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), andGSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate for anenzyme selected from the group consisting of those shown in Table 3. Insome embodiments, the activatable antibody in an uncleaved stateincludes a spacer, and the spacer is joined directly to the MM and hasthe structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.

The invention provides methods of selectively conjugating an agent to anactivatable antibody. For example, the invention provides a method ofpartially reducing and conjugating an agent to an activatable antibodyresulting in selectivity in the placement of the agent by partiallyreducing at least one interchain disulfide bond in the activatableantibody with a reducing agent without disturbing any intrachaindisulfide bonds in the activatable antibody, and conjugating the agentto at least one interchain thiol, wherein the activatable antibodyincludes an antibody or an antigen binding fragment thereof (AB) thatspecifically binds to a target, a masking moiety (MM) that inhibits thebinding of the AB of the activatable antibody in an uncleaved state tothe target, and a cleavable moiety (CM) coupled to the AB, and the CM isa polypeptide that functions as a substrate for a protease.

The invention also provides a method of partially reducing andconjugating an agent to an activatable antibody resulting in selectivityin the placement of the agent by partially reducing at least oneinterchain disulfide bond in the activatable antibody with a reducingagent without disturbing or otherwise compromising the activity and/orefficacy of the activatable antibody, wherein the activatable antibodyincludes an antibody or an antigen binding fragment thereof (AB) thatspecifically binds to a target, a masking moiety (MM) that inhibits thebinding of the AB of the activatable antibody in an uncleaved state tothe target, and a cleavable moiety (CM) coupled to the AB, and the CM isa polypeptide that functions as a substrate for a protease. The activityand/or efficacy of the activatable antibody is, by way of nonlimitingexample, masking activity, activation of the activatable antibody,and/or binding activity of the activated activatable antibody.

In some embodiments, the method does not disturb one or more intrachaindisulfide bonds of the activatable antibody. In some embodiments, themethod does not disturb one or more intrachain disulfide bonds of the MMwithin the activatable antibody.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP. Insome embodiments, the agent is selected from the group of agents listedin Table 4. In some embodiments, the agent is a toxin or fragmentthereof. In some embodiments, the agent is a microtubule inhibitor. Insome embodiments, the agent is a dolastatin. In some embodiments, theagent is an auristatin or derivative thereof. In some embodiments, theagent is auristatin E or a derivative thereof. In some embodiments, theagent is monomethyl auristatin E (MMAE). In some embodiments, the agentis monomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or a derivative thereof. In some embodiments, the agent isDM1 or DM4. In some embodiments, the agent is a nucleic acid damagingagent. In some embodiments, the agent is a duocarmycin or derivativethereof. In some embodiments, the agent is a calicheamicin or derivativethereof. In some embodiments, the agent is conjugated to the AB via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the agent is a detectable moiety. In some embodiments, thedetectable moiety is a diagnostic agent.

In some embodiments, the target is selected from the group of targetslisted in Table 1. In some embodiments, the AB is or is derived from anantibody selected from the group of antibodies listed in Table 2. Insome embodiments, the antigen binding fragment thereof is selected fromthe group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, ascAb, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, the AB has an equilibriumdissociation constant of about 100 nM or less for binding to the target.In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to the target. In some embodiments, the MM isa polypeptide of no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget, and the MM polypeptide sequence is no more than 50% identical toany natural binding partner of the AB. In some embodiments, the MM doesnot include more than 25% amino acid sequence identity to the target. Insome embodiments, the MM does not include more than 10% amino acidsequence identity to the target. In some embodiments, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments, theprotease is co-localized with the target in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the activatableantibody includes a linking peptide between the MM and the CM. In someembodiments, the activatable antibody includes a linking peptide betweenthe CM and the AB. In some embodiments, the activatable antibodyincludes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in an uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, at least one of LP1 or LP2includes an amino acid sequence selected from the group consisting of(GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 21) and (GGGS)_(n)(SEQ IDNO: 22), where n is an integer of at least one. In some embodiments, atleast one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24),GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), andGSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate for anenzyme selected from the group consisting of those shown in Table 3. Insome embodiments, the activatable antibody in an uncleaved stateincludes a spacer, and the spacer is joined directly to the MM and hasthe structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.

The invention provides a method of partially reducing and conjugating anagent to an activatable antibody resulting in selectivity in theplacement of the agent by partially reducing at least one disulfide bondbetween the activatable antibody and a second molecule with a reducingagent without disturbing any intrachain disulfide bonds in theactivatable antibody, and conjugating the agent to at least one thiol,wherein the activatable antibody includes an antibody or an antigenbinding fragment thereof (AB) that specifically binds to a target, amasking moiety (MM) that inhibits the binding of the AB of theactivatable antibody in an uncleaved state to the target, and acleavable moiety (CM) coupled to the AB, and the CM is a polypeptidethat functions as a substrate for a protease. In some embodiments, thesecond molecule is cysteine. In some embodiments, the second molecule isglutathione.

The invention also provides a method of partially reducing andconjugating an agent to an activatable antibody resulting in selectivityin the placement of the agent by partially reducing at least onedisulfide bond between the activatable antibody and a second moleculewith a reducing agent without disturbing or otherwise compromising theactivity and/or efficacy of the activatable antibody, wherein theactivatable antibody includes an antibody or an antigen binding fragmentthereof (AB) that specifically binds to a target, a masking moiety (MM)that inhibits the binding of the AB of the activatable antibody in anuncleaved state to the target, and a cleavable moiety (CM) coupled tothe AB, and the CM is a polypeptide that functions as a substrate for aprotease. The activity and/or efficacy of the activatable antibody is,by way of nonlimiting example, masking activity, activation of theactivatable antibody, and/or binding activity of the activatedactivatable antibody. In some embodiments, the second molecule iscysteine. In some embodiments, the second molecule is glutathione.

In some embodiments, the method does not disturb one or more intrachaindisulfide bonds of the activatable antibody. In some embodiments, themethod does not disturb one or more intrachain disulfide bonds of the MMwithin the activatable antibody.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP. Insome embodiments, the agent is selected from the group of agents listedin Table 4. In some embodiments, the agent is a toxin or fragmentthereof. In some embodiments, the agent is a microtubule inhibitor. Insome embodiments, the agent is a dolastatin. In some embodiments, theagent is an auristatin or derivative thereof. In some embodiments, theagent is auristatin E or a derivative thereof. In some embodiments, theagent is monomethyl auristatin E (MMAE). In some embodiments, the agentis monomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or a derivative thereof. In some embodiments, the agent isDM1 or DM4. In some embodiments, the agent is a nucleic acid damagingagent. In some embodiments, the agent is a duocarmycin or derivativethereof. In some embodiments, the agent is a calicheamicin or derivativethereof. In some embodiments, the agent is conjugated to the AB via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the agent is a detectable moiety. In some embodiments, thedetectable moiety is a diagnostic agent.

In some embodiments, the target is selected from the group of targetslisted in Table 1. In some embodiments, the AB is or is derived from anantibody selected from the group of antibodies listed in Table 2. Insome embodiments, the antigen binding fragment thereof is selected fromthe group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, ascAb, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, the AB has an equilibriumdissociation constant of about 100 nM or less for binding to the target.In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to the target. In some embodiments, the MM isa polypeptide of no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget, and the MM polypeptide sequence is no more than 50% identical toany natural binding partner of the AB. In some embodiments, the MM doesnot include more than 25% amino acid sequence identity to the target. Insome embodiments, the MM does not include more than 10% amino acidsequence identity to the target. In some embodiments, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments, theprotease is co-localized with the target in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the activatableantibody includes a linking peptide between the MM and the CM. In someembodiments, the activatable antibody includes a linking peptide betweenthe CM and the AB. In some embodiments, the activatable antibodyincludes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in an uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, at least one of LP1 or LP2includes an amino acid sequence selected from the group consisting of(GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 21) and (GGGS)_(n)(SEQ IDNO: 22), where n is an integer of at least one. In some embodiments, atleast one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24),GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), andGSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate for anenzyme selected from the group consisting of those shown in Table 3. Insome embodiments, the activatable antibody in an uncleaved stateincludes a spacer, and the spacer is joined directly to the MM and hasthe structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.

The invention also provides a method of partially reducing anactivatable antibody resulting in selectivity in the placement of one ormore potential conjugation sites in the activatable antibody bypartially reducing at least one interchain disulfide bond in theactivatable antibody with a reducing agent without disturbing anyintrachain disulfide bonds in the activatable antibody, wherein theactivatable antibody includes an antibody or an antigen binding fragmentthereof (AB) that specifically binds to a target, a masking moiety (MM)that inhibits the binding of the AB of the activatable antibody in anuncleaved state to the target, and a cleavable moiety (CM) coupled tothe AB, and the CM is a polypeptide that functions as a substrate for aprotease.

The invention also provides a method of partially reducing anactivatable antibody resulting in selectivity in the placement of one ormore potential conjugation sites in the activatable antibody bypartially reducing at least one interchain disulfide bond in theactivatable antibody with a reducing agent without disturbing orotherwise compromising the activity and/or efficacy of the activatableantibody, wherein the activatable antibody includes an antibody or anantigen binding fragment thereof (AB) that specifically binds to atarget, a masking moiety (MM) that inhibits the binding of the AB of theactivatable antibody in an uncleaved state to the target, and acleavable moiety (CM) coupled to the AB, and the CM is a polypeptidethat functions as a substrate for a protease. The activity and/orefficacy of the activatable antibody is, by way of nonlimiting example,masking activity, activation of the activatable antibody, and/or bindingactivity of the activated activatable antibody.

In some embodiments, the method does not disturb one or more intrachaindisulfide bonds of the activatable antibody. In some embodiments, themethod does not disturb one or more intrachain disulfide bonds of the MMwithin the activatable antibody.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP.

In some embodiments, the target is selected from the group of targetslisted in Table 1. In some embodiments, the AB is or is derived from anantibody selected from the group of antibodies listed in Table 2. Insome embodiments, the antigen binding fragment thereof is selected fromthe group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, ascAb, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, the AB has an equilibriumdissociation constant of about 100 nM or less for binding to the target.In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to the target. In some embodiments, the MM isa polypeptide of no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget, and the MM polypeptide sequence is no more than 50% identical toany natural binding partner of the AB. In some embodiments, the MM doesnot include more than 25% amino acid sequence identity to the target. Insome embodiments, the MM does not include more than 10% amino acidsequence identity to the target. In some embodiments, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments, theprotease is co-localized with the target in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the activatableantibody includes a linking peptide between the MM and the CM. In someembodiments, the activatable antibody includes a linking peptide betweenthe CM and the AB. In some embodiments, the activatable antibodyincludes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in an uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, at least one of LP1 or LP2includes an amino acid sequence selected from the group consisting of(GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 21) and (GGGS)_(n)(SEQ IDNO: 22), where n is an integer of at least one. In some embodiments, atleast one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24),GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), andGSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate for anenzyme selected from the group consisting of those shown in Table 3. Insome embodiments, the activatable antibody in an uncleaved stateincludes a spacer, and the spacer is joined directly to the MM and hasthe structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.

The invention also provides a method of partially reducing anactivatable antibody resulting in selectivity in the placement of one ormore potential conjugation sites in the activatable antibody bypartially reducing at least one disulfide bond between the activatableantibody and a second molecule with a reducing agent without disturbingany intrachain disulfide bonds in the activatable antibody, wherein theactivatable antibody includes an antibody or an antigen binding fragmentthereof (AB) that specifically binds to a target, a masking moiety (MM)that inhibits the binding of the AB of the activatable antibody in anuncleaved state to the target, and a cleavable moiety (CM) coupled tothe AB, and the CM is a polypeptide that functions as a substrate for aprotease. In some embodiments, the second molecule is cysteine. In someembodiments, the second molecule is glutathione.

The invention also provides a method of partially reducing anactivatable antibody resulting in selectivity in the placement of one ormore potential conjugation sites in the activatable antibody bypartially reducing at least one disulfide bond between the activatableantibody and a second molecule with a reducing agent without disturbingor otherwise compromising the activity and/or efficacy of theactivatable antibody, wherein the activatable antibody includes anantibody or an antigen binding fragment thereof (AB) that specificallybinds to a target, a masking moiety (MM) that inhibits the binding ofthe AB of the activatable antibody in an uncleaved state to the target,and a cleavable moiety (CM) coupled to the AB, and the CM is apolypeptide that functions as a substrate for a protease. The activityand/or efficacy of the activatable antibody is, by way of nonlimitingexample, masking activity, activation of the activatable antibody,and/or binding activity of the activated activatable antibody. In someembodiments, the second molecule is cysteine. In some embodiments, thesecond molecule is glutathione.

In some embodiments, the method does not disturb one or more intrachaindisulfide bonds of the activatable antibody. In some embodiments, themethod does not disturb one or more intrachain disulfide bonds of the MMwithin the activatable antibody.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, reducing agent is TCEP. Insome embodiments, the agent is selected from the group of agents listedin Table 4. In some embodiments, the agent is a toxin or fragmentthereof. In some embodiments, the agent is a microtubule inhibitor. Insome embodiments, the agent is a dolastatin. In some embodiments, theagent is an auristatin or derivative thereof. In some embodiments, theagent is auristatin E or a derivative thereof. In some embodiments, theagent is monomethyl auristatin E (MMAE). In some embodiments, the agentis monomethyl auristatin D (MMAD). In some embodiments, the agent is amaytansinoid or a derivative thereof. In some embodiments, the agent isDM1 or DM4. In some embodiments, the agent is a nucleic acid damagingagent. In some embodiments, the agent is a duocarmycin or derivativethereof. In some embodiments, the agent is a calicheamicin or derivativethereof. In some embodiments, the agent is conjugated to the AB via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the agent is a detectable moiety. In some embodiments, thedetectable moiety is a diagnostic agent.

In some embodiments, the target is selected from the group of targetslisted in Table 1. In some embodiments, the AB is or is derived from anantibody selected from the group of antibodies listed in Table 2. Insome embodiments, the antigen binding fragment thereof is selected fromthe group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, ascAb, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, the AB has an equilibriumdissociation constant of about 100 nM or less for binding to the target.In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to the target. In some embodiments, the MM isa polypeptide of no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget, and the MM polypeptide sequence is no more than 50% identical toany natural binding partner of the AB. In some embodiments, the MM doesnot include more than 25% amino acid sequence identity to the target. Insome embodiments, the MM does not include more than 10% amino acidsequence identity to the target. In some embodiments, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments, theprotease is co-localized with the target in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the activatableantibody includes a linking peptide between the MM and the CM. In someembodiments, the activatable antibody includes a linking peptide betweenthe CM and the AB. In some embodiments, the activatable antibodyincludes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in an uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, at least one of LP1 or LP2includes an amino acid sequence selected from the group consisting of(GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 21) and (GGGS)_(n)(SEQ IDNO: 22), where n is an integer of at least one. In some embodiments, atleast one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24),GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), andGSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate for anenzyme selected from the group consisting of those shown in Table 3. Insome embodiments, the activatable antibody in an uncleaved stateincludes a spacer, and the spacer is joined directly to the MM and hasthe structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.

The invention also provides conjugated activatable antibodies thatinclude an activatable antibody linked to monomethyl auristatin D (MMAD)payload, wherein the activatable antibody includes an antibody or anantigen binding fragment thereof (AB) that specifically binds to atarget, a masking moiety (MM) that inhibits the binding of the AB of theactivatable antibody in an uncleaved state to the target, and acleavable moiety (CM) coupled to the AB, and the CM is a polypeptidethat functions as a substrate for a protease.

In some embodiments, the MMAD-conjugated activatable antibody can beconjugated using any of several methods for attaching agents to ABs: (a)attachment to the carbohydrate moieties of the AB, or (b) attachment tosulfhydryl groups of the AB, or (c) attachment to amino groups of theAB, or (d) attachment to carboxylate groups of the AB.

In some embodiments, the MMAD payload is conjugated to the AB via alinker. In some embodiments, the MMAD payload is conjugated to acysteine in the AB via a linker. In some embodiments, the MMAD payloadis conjugated to a lysine in the AB via a linker. In some embodiments,the MMAD payload is conjugated to another residue of the AB via alinker, such as those residues disclosed herein. In some embodiments,the linker is a thiol-containing linker. In some embodiments, the linkeris a cleavable linker. In some embodiments, the linker is anon-cleavable linker. In some embodiments, the linker is selected fromthe group consisting of the linkers shown in Tables 5 and 6. In someembodiments, the activatable antibody and the MMAD payload are linkedvia a maleimide caproyl-valine-citrulline linker. In some embodiments,the activatable antibody and the MMAD payload are linked via a maleimidePEG-valine-citrulline linker. In some embodiments, the activatableantibody and the MMAD payload are linked via a maleimidecaproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In someembodiments, the activatable antibody and the MMAD payload are linkedvia a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyllinker. In some embodiments, the MMAD payload is conjugated to the ABusing the partial reduction and conjugation technology disclosed herein.

In some embodiments, the target is selected from the group of targetslisted in Table 1. In some embodiments, the target is EGFR. In someembodiments, the target is a Jagged protein, e.g., Jagged 1 and/orJagged 2. In some embodiments, the target is interleukin 6 receptor(IL-6R). In some embodiments, the AB is or is derived from an antibodyselected from the group of antibodies listed in Table 2. In someembodiments, the antigen binding fragment thereof is selected from thegroup consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb,a dAb, a single domain heavy chain antibody, and a single domain lightchain antibody. In some embodiments, the AB has an equilibriumdissociation constant of about 100 nM or less for binding to the target.In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB that is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to the target. In some embodiments, the MM isa polypeptide of no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget, and the MM polypeptide sequence is no more than 50% identical toany natural binding partner of the AB. In some embodiments, the MM doesnot include more than 25% amino acid sequence identity to the target. Insome embodiments, the MM does not include more than 10% amino acidsequence identity to the target. In some embodiments, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments, theprotease is co-localized with the target in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the activatableantibody includes a linking peptide between the MM and the CM. In someembodiments, the activatable antibody includes a linking peptide betweenthe CM and the AB. In some embodiments, the activatable antibodyincludes a first linking peptide (LP1) and a second linking peptide(LP2), and the activatable antibody in an uncleaved state has thestructural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length. In some embodiments, at least one of LP1 or LP2includes an amino acid sequence selected from the group consisting of(GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 21) and (GGGS)_(n) (SEQ IDNO: 22), where n is an integer of at least one. In some embodiments, atleast one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24),GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), andGSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate for anenzyme selected from the group consisting of those shown in Table 3. Insome embodiments, the activatable antibody in an uncleaved stateincludes a spacer, and the spacer is joined directly to the MM and hasthe structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.

The invention provides methods of treating, preventing and/or delayingthe onset or progression of, or alleviating a symptom of an indication,e.g., disease or disorder, associated with expression and/or activity ofthe target in a subject using a conjugated activatable antibody that inan activated state binds the target, particularly a conjugatedactivatable antibody that binds and neutralizes or otherwise inhibits atleast one biological activity of the target. Suitable conjugatedactivatable antibodies for use in any of the methods and kits of theinvention include any of the conjugated activatable antibodies describedherein, including any partially conjugated activatable antibodies and/orpartially reduced activatable antibodies described herein.

In some embodiments, the invention provides methods of treating,preventing and/or delaying the onset or progression of, or alleviating asymptom of an indication, e.g., disease or disorder, associated with adetectable level of expression and/or activity of the target in asubject using a conjugated activatable antibody that in an activatedstate binds the target, particularly a conjugated activatable antibodythat binds and neutralizes or otherwise inhibits at least one biologicalactivity of the target. In some embodiments, the conjugated activatableantibody in an activated state binds the target and is internalized. Insome embodiments, the detectable level of expression and/or activity ofthe target is found in at least one intended site of therapy and/ordiagnosis. In some embodiments, the detectable level of expressionand/or activity of the target is found in normal, e.g., healthy, tissue,and the conjugated activatable antibody is activated at the intendedsite(s) of therapy and/or diagnosis but not in the normal, e.g.,healthy, tissue. The conjugated activatable antibody is activated, forexample, by a protease that is co-localized with the target at theintended site(s) of therapy and/or diagnosis. In some embodiments, thedetectable level of expression and/or activity of the target is found inat least one intended site of therapy and/or diagnosis and in normal,e.g., healthy, tissue, and the conjugated activatable antibody isactivated at the intended site(s) of therapy and/or diagnosis but not inthe normal, e.g., healthy, tissue. The conjugated activatable antibodyis activated, for example, by a protease that is co-localized with thetarget at the intended site(s) of therapy and/or diagnosis.

In some embodiments, the indication, e.g., disease or disorder,associated with expression and/or activity of the target is a cancer. Insome embodiments, the indication, e.g., disease or disorder, associatedwith expression and/or activity of the target is an inflammatorydisorder and/or an autoimmune disease.

The invention also provides methods of inhibiting angiogenesis in asubject by administering a therapeutically effective amount of aconjugated activatable antibody described herein to a subject in needthereof.

The conjugated activatable antibody can be administered at any stage ofthe disease. In some embodiments, a conjugated activatable antibody canbe administered to a patient suffering cancer of any stage, from earlyto metastatic. In some embodiments, a conjugated activatable antibodycan be administered to a patient suffering from an inflammatory disorderand/or autoimmune disease of any stage, from early onset to an advancedstage. It is to be understood that the terms subject and patient areused interchangeably herein.

The conjugated activatable antibodies are also useful in othertherapeutic indications and treatment regimens. For example, theconjugated activatable antibodies of the embodiments provided herein canbe used in a treatment regimen that includes neoadjuvant therapy.

In some embodiments, a conjugated activatable antibody is administeredin combination with one or more additional agents such as, by way ofnon-limiting example, a chemotherapeutic agent, such as an alkylatingagent, an anti-metabolite, an anti-microtubule agent, a topoisomeraseinhibitor, a cytotoxic antibiotic, and any other nucleic acid damagingagent. In some embodiments, the additional agent is a taxane, such aspaclitaxel (e.g., Abraxane®). In some embodiments, the additional agentis an anti-metabolite, such as gemcitabine. In some embodiments, theadditional agent is an alkylating agent, such as platinum-basedchemotherapy, such as carboplatin or cisplatin. In some embodiments, theadditional agent is a targeted agent, such as a kinase inhibitor, e.g.,sorafenib or erlotinib. In some embodiments, the additional agent is atargeted agent, such as another antibody, e.g., a monoclonal antibody(e.g., bevacizumab), a bispecific antibody, or a multispecific antibody.In some embodiments, the additional agent is a proteosome inhibitor,such as bortezomib or carfilzomib. In some embodiments, the additionalagent is an immune modulating agent, such as lenolidominde or IL-2. Insome embodiments, the additional agent is radiation. In someembodiments, the additional agent is an agent considered standard ofcare by those skilled in the art. In some embodiments, the additionalagent is a chemotherapeutic agent well known to those skilled in theart. In some embodiments, the conjugated activatable antibody and theadditional agent(s) are formulated in a single composition. In someembodiments, the conjugated activatable antibody and the additionalagent(s) are administered as two or more separate compositions. In someembodiments, the conjugated activatable antibody and the additionalagent(s) are administered simultaneously. In some embodiments, theconjugated activatable antibody and the additional agent(s) areadministered sequentially.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is a non-humanmammal, such as a non-human primate, companion animal (e.g., cat, dog,horse), farm animal, work animal, or zoo animal. In some embodiments,the subject is a rodent. In some embodiments, the subject is a human. Insome embodiments, the subject is a companion animal. In someembodiments, the subject is an animal in the care of a veterinarian.

The conjugated activatable antibody and therapeutic formulations thereofare administered to a subject suffering from or susceptible to a diseaseor disorder associated with expression and/or activity of the target. Asubject suffering from or susceptible to a disease or disorderassociated with expression and/or activity of the target is identifiedusing any of a variety of methods known in the art. For example,subjects suffering from cancer or other neoplastic condition areidentified using any of a variety of clinical and/or laboratory testssuch as, physical examination and blood, urine and stool analysis toevaluate health status.

Administration of a conjugated activatable antibody to a patientsuffering from a disease or disorder associated with target expressionand/or activity is considered successful if any of a variety oflaboratory or clinical objectives is achieved. For example,administration of a conjugated activatable antibody to a patientsuffering from a disease or disorder associated with target expressionand/or activity is considered successful if one or more of the symptomsassociated with the disease or disorder is alleviated, reduced,inhibited or does not progress to a further, i.e., worse, state.Administration of a conjugated activatable antibody to a patientsuffering from a disease or disorder associated with target expressionand/or activity is considered successful if the disease enters remissionor does not progress to a further, i.e., worse, state.

The invention also provides methods of using conjugated activatableantibodies that bind the target in a variety of diagnostic and/orprophylactic indications, as well as kits for use in these methods. Insome embodiments of these methods and/or kits, the conjugatedactivatable antibody includes a detectable label. In some embodiments ofthese methods and/or kits, the detectable label includes an imagingagent, a contrasting agent, an enzyme, a fluorescent label, achromophore, a dye, one or more metal ions, or a ligand-based label. Insome embodiments of these methods and/or kits, the imaging agentcomprises a radioisotope. In some embodiments of these methods, theradioisotope is indium or technetium. In some embodiments of thesemethods, the radioisotope is or is derived from iodine. In someembodiments of these methods, the radioisotope is ¹²⁵I or ¹³³I. In someembodiments of these methods and/or kits, the contrasting agentcomprises iodine, gadolinium or iron oxide. In some embodiments of thesemethods and/or kits, the enzyme comprises horseradish peroxidase,alkaline phosphatase, or β-galactosidase. In some embodiments of thesemethods and/or kits, the fluorescent label comprises yellow fluorescentprotein (YFP), cyan fluorescent protein (CFP), green fluorescent protein(GFP), modified red fluorescent protein (mRFP), red fluorescent proteintdimer2 (RFP tdimer2), HCRED, or a europium derivative. In someembodiments of these methods and/or kits, the luminescent labelcomprises an N-methylacrydium derivative. In some embodiments of thesemethods and/or kits, the label comprises an Alexa Fluor® label, such asAlex Fluor® 680 or Alexa Fluor® 750. In some embodiments of thesemethods and/or kits, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments of these methods and/or kits, the subject is amammal. In some embodiments of these methods and/or kits, the subject isa human. In some embodiments, the subject is a non-human mammal, such asa non-human primate, companion animal (e.g., cat, dog, horse), farmanimal, work animal, or zoo animal. In some embodiments, the subject isa rodent. In some embodiments, the subject is a human. In someembodiments, the subject is a companion animal. In some embodiments, thesubject is an animal in the care of a veterinarian.

The invention also provides methods of using the conjugated activatableantibodies (i.e., activatable antibody conjugates) in a variety ofdiagnostic and/or prophylactic indications. For example, the inventionprovides methods of detecting presence or absence of a cleaving agentand a target of interest in a subject or a sample by (i) contacting asubject or sample with a conjugated activatable antibody and (ii)measuring a level of conjugated activatable antibody in the subject orsample, wherein a detectable level of activated conjugated activatableantibody in the subject or sample indicates that the cleaving agent andthe target are present in the subject or sample and wherein nodetectable level of activated conjugated activatable antibody in thesubject or sample indicates that the cleaving agent, the target or boththe cleaving agent and the target are absent in the subject or sample.In some embodiments an unconjugated activatable antibody correspondingto the activatable antibody conjugated in the conjugated activatableantibody is used to contact the subject or sample.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with a conjugated activatable antibody in the presence of thetarget, and (ii) measuring a level of activated conjugated activatableantibody in the subject or sample, wherein a detectable level ofactivated conjugated activatable antibody in the subject or sampleindicates that the cleaving agent is present in the subject or sampleand wherein no detectable level of conjugated activatable antibody inthe subject or sample indicates that the cleaving agent is absent in thesubject or sample. In some embodiments an unconjugated activatableantibody corresponding to the activatable antibody conjugated in theconjugated activatable antibody is used to contact the subject orsample.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with a conjugated activatable antibody; and (ii) measuring alevel of detectable label in the subject or sample, wherein a detectablelevel of the detectable label in the subject or sample indicates thatthe cleaving agent is absent in the subject or sample and wherein nodetectable level of the detectable label in the subject or sampleindicates that the cleaving agent is present in the subject or sample.In some embodiments an unconjugated activatable antibody correspondingto the activatable antibody conjugated in the conjugated activatableantibody is used to contact the subject or sample.

In some embodiments of these methods, the conjugated activatableantibody or corresponding unconjugated activatable antibody includes adetectable label selected from the group consisting of an imaging agent,a contrasting agent, an enzyme, a fluorescent label, a chromophore, adye, one or more metal ions, and a ligand-based label. In someembodiments of these methods, the imaging agent comprises aradioisotope. In some embodiments of these methods, the radioisotope isindium or technetium. In some embodiments of these methods, thecontrasting agent comprises iodine, gadolinium or iron oxide. In someembodiments of these methods, the enzyme comprises horseradishperoxidase, alkaline phosphatase, or 3-galactosidase. In someembodiments of these methods, the fluorescent label comprises yellowfluorescent protein (YFP), cyan fluorescent protein (CFP), greenfluorescent protein (GFP), modified red fluorescent protein (mRFP), redfluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europiumderivative. In some embodiments of these methods, the luminescent labelcomprises an N-methylacrydium derivative. In some embodiments of thesemethods, the label comprises an Alexa Fluor® label, such as Alex Fluor®680 or Alexa Fluor® 750. In some embodiments of these methods, theligand-based label comprises biotin, avidin, streptavidin or one or morehaptens.

In some embodiments of these methods, the subject is a mammal. In someembodiments of these methods, the subject is a human. In someembodiments, the subject is a non-human mammal, such as a non-humanprimate, companion animal (e.g., cat, dog, horse), farm animal, workanimal, or zoo animal. In some embodiments, the subject is a rodent. Insome embodiments, the subject is a human. In some embodiments, thesubject is a companion animal. In some embodiments, the subject is ananimal in the care of a veterinarian.

In some embodiments of these methods, the method is an in vivo method.In some embodiments of these methods, the method is an in situ method.In some embodiments of these methods, the method is an ex vivo method.In some embodiments of these methods, the method is an in vitro method.

In some embodiments of the methods, the method is used to identify orotherwise refine a patient population suitable for treatment with aconjugated activatable antibody of the disclosure. For example, patientsthat test positive for both the target and a protease that cleaves thesubstrate in the cleavable moiety (CM) of the conjugated activatableantibody being tested in these methods are identified as suitablecandidates for treatment with such a conjugated activatable antibodycomprising such a CM. Likewise, patients that test negative for eitheror both of the target and the protease that cleaves the substrate in theCM in the conjugated activatable antibody or corresponding unconjugatedactivatable antibody being tested using these methods might beidentified as suitable candidates for another form of therapy. In someembodiments, such patients can be tested with other activatableantibodies and/or conjugated activatable antibodies until a suitableconjugated activatable antibody for treatment is identified (e.g., aconjugated activatable antibody comprising a CM that is cleaved by thepatient at the site of disease).

In some embodiments of the methods, the method is used to identify orotherwise refine a patient population suitable for treatment with aconjugated activatable antibody of the disclosure followed by treatmentby administering that conjugated activatable antibody to a subject inneed thereof. For example, patients that test positive for both thetarget and a protease that cleaves the substrate in the cleavable moiety(CM) of the conjugated activatable antibody or correspondingunconjugated activatable antibody being tested in these methods areidentified as suitable candidates for treatment with such a conjugatedactivatable antibody comprising such a CM, and the patient is thenadministered a therapeutically effective amount of the conjugatedactivatable antibody. Likewise, patients that test negative for eitheror both of the target and the protease that cleaves the substrate in theCM in the conjugated activatable antibody or corresponding unconjugatedactivatable antibody being tested using these methods might beidentified as suitable candidates for another form of therapy. In someembodiments, such patients can be tested with other activatableantibodies and/or conjugated activatable antibodies until a conjugatedactivatable antibody for treatment is identified (e.g., a conjugatedactivatable antibody comprising a CM that is cleaved by the patient atthe site of disease). In some embodiments, the patient is thenadministered a therapeutically effective amount of the conjugatedactivatable antibody for which the patient tested positive.

The invention also provides conjugated activatable antibodies that in anactivated state bind a target, wherein the conjugated activatableantibody includes an antibody or an antigen binding fragment thereof(AB) that specifically binds to the target, wherein the AB is conjugatedto monomethyl auristatin D (MMAD); a masking moiety (MM) that inhibitsthe binding of the AB to the target when the activatable antibody is inan uncleaved state; and a cleavable moiety (CM) coupled to the AB,wherein the CM is a polypeptide that functions as a substrate for aprotease.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM. In some embodiments, the activatable antibodycomprises a linking peptide between the MM and the CM. In someembodiments, the activatable antibody comprises a linking peptidebetween the CM and the AB. In some embodiments, the activatable antibodycomprises a first linking peptide (LP1) and a second linking peptide(LP2), and wherein the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other. In someembodiments, each of LP1 and LP2 is a peptide of about 1 to 20 aminoacids in length.

In some embodiments, he MM has an equilibrium dissociation constant forbinding to the AB which is greater than the equilibrium dissociationconstant of the AB to the target. In some embodiments, the MM does notinterfere or compete with the AB for binding to the target when theactivatable antibody is in a cleaved state. In some embodiments, the MMis a polypeptide of about no more than 40 amino acids in length. In someembodiments, the MM polypeptide sequence is different from that of thetarget and wherein the MM polypeptide sequence is no more than 50%identical to any natural binding partner of the AB.

In some embodiments, the protease is co-localized with the target in atissue, and wherein the protease cleaves the CM in the activatableantibody when the activatable antibody is exposed to the protease. Insome embodiments, the CM is a polypeptide of up to 15 amino acids inlength. In some embodiments, the CM is a substrate for an enzymeselected from the group consisting of those shown in Table 3.

In some embodiments, the antigen binding fragment thereof is selectedfrom the group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv,a scab, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody. In some embodiments, the target for the AB isselected from the group consisting of the targets listed in Table 1. Insome embodiments, the AB is or is derived from an antibody listed inTable 2.

In some embodiments, the MMAD is conjugated to the AB via a linker. Insome embodiments, linker is a cleavable linker. In some embodiments, thelinker is a non-cleavable linker. In some embodiments, the linker isselected from the group consisting of the linkers shown in Tables 5 and6. In some embodiments, the activatable antibody and the MMAD payloadare linked via a maleimide caproyl-valine-citrulline linker. In someembodiments, the activatable antibody and the MMAD payload are linkedvia a maleimide PEG-valine-citrulline linker. In some embodiments, theactivatable antibody and the MMAD payload are linked via a maleimidecaproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In someembodiments, the activatable antibody and the MMAD payload are linkedvia a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyllinker. In some embodiments, the MMAD payload is conjugated to the ABusing the partial reduction and conjugation technology disclosed herein.

Pharmaceutical compositions according to the invention can include aconjugated antibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits for use in the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph demonstrating that partial reduction of an activatableanti-EGFR antibody using three different TCEP-to-activatable antibodyratios (i.e., ratios of 1.5:1, of 2:1, and of 4:1), and subsequent thiolconjugation of a fluorescent dye (Alexa 680) to such partially reducedactivatable anti-EGFR antibody with a cleavable moiety(3954-1204-c225v5) using the methods provided herein successfullyconjugates the dye to the activatable antibody, while maintaining themasking efficiency of the masking moiety of the activatable anti-EGFRantibody. As used in these figures, “(1.5)”, “(2)” and “(4)” signify theratios of TCEP-to-activatable antibody used in the TCEP reduction step.

FIG. 2 is a graph demonstrating that partial reduction of a maskedanti-EGFR antibody with a noncleavable moiety using three differentTCEP-to-masked antibody ratios (i.e., ratios of 1.5:1, of 2:1, and of4:1), and subsequent thiol conjugation of Alexa 680 to such partiallyreduced masked anti-EGFR antibody with a noncleavable moiety(3954-NSUB-c225v5) using the methods provided herein successfullyconjugates the dye to the noncleavable masked antibody, whilemaintaining the masking efficiency of the masking moiety of the maskedanti-EGFR antibody. As used in these figures, “(1.5)”, “(2)” and “(4)”signify the ratios of TCEP-to-masked antibody used in the TCEP reductionstep.

FIGS. 3A and 3B are an illustration and a graph demonstrating thenon-reduced analysis by LabChip® of TCEP reduction of 3954-1204-c225v5,Alexa 680 thiol-conjugation and activation by the protease uPA. As shownin these figures using two different TCEP-to-activatable antibody ratios(i.e., ratios of 1.5:1 and of 4:1), partial reduction, subsequent thiolconjugation of Alexa 680 to the partially reduced activatable anti-EGFRantibody 3954-1204-c225v5 and activation by uPA does not disturb orotherwise negatively affect the activation and/or masking efficiency ofthe activatable antibody. As used in these figures, “1.5” and “4”signify the ratios of TCEP-to-activatable antibody used in the TCEPreduction step; and “(U)” signifies that the activatable antibody hasbeen activated, i.e., cleaved, by incubation with uPA.

FIGS. 4A and 4B are an illustration and a graph demonstrating thereduced analysis by LabChip® of TCEP reduction of 3954-1204-c225v5,Alexa 680 thiol-conjugation and activation by the protease uPA. As shownin these figures using two different TCEP-to-activatable antibody ratios(i.e., ratios of 1.5:1 and of 4:1), partial reduction, subsequent thiolconjugation of Alexa 680 to the partially reduced activatable anti-EGFRantibody 3954-1204-c225v5 and activation by uPA does not disturb orotherwise negatively affect the activation and/or masking efficiency ofthe activatable antibody. As used in these figures, “1.5” and “4”signify the ratios of TCEP-to-activatable antibody used in the TCEPreduction step; “(U)” signifies that the activatable antibody has beenactivated, i.e., cleaved, by incubation with uPA, and “R” signifiesreduced analysis.

FIGS. 5A and 5B are an illustration and a graph demonstrating thenon-reduced analysis by LabChip® of TCEP reduction of 3954-NSUB-c225v5,Alexa 680 thiol-conjugation and activation by the protease uPA. As shownin these figures using two different TCEP-to-masked antibody ratios(i.e., ratios of 1.5:1 and of 4:1), partial reduction, subsequent thiolconjugation of Alexa 680 to the partially reduced masked anti-EGFRantibody 3954-NSUB-c225v5 and activation by uPA does not disturb orotherwise negatively affect the activation and/or masking efficiency ofthe noncleavable masked antibody. As used in these figures, “1.5” and“4” signify the ratios of TCEP-to-masked antibody used in the TCEPreduction step; and “(U)” signifies that the noncleavable maskedantibody was not activated, i.e., not cleaved, by incubation with uPA.

FIGS. 6A and 6B are an illustration and a graph demonstrating thereduced analysis by LabChip® of TCEP reduction of 3954-NSUB-c225v5,Alexa 680 thiol-conjugation and activation by the protease uPA. As shownin these figures using two different TCEP-to-masked antibody ratios(i.e., ratios of 1.5:1 and of 4:1), partial reduction, subsequent thiolconjugation of Alexa 680 to the partially reduced masked anti-EGFRantibody 3954-NSUB-c225v5 and activation by uPA does not disturb orotherwise negatively affect the activation and/or masking efficiency ofthe noncleavable masked antibody. As used in these figures, “1.5” and“4” signify the ratios of TCEP-to-masked antibody used in the TCEPreduction step; “(U)” signifies that the noncleavable masked antibodywas not been activated, i.e., not cleaved by incubation with uPA, and“R” signifies reduced analysis.

FIG. 7 is a table and a photograph depicting reduction of theactivatable anti-Jagged antibody 5342-1204-4D11 at a ratio of TCEP toactivatable antibody equaling 4:1 using a 120-minute reduction time. Forthe data shown, reduction was followed by conjugation to a fluorescentdye, Alexa 680.

FIG. 8 is a graph depicting varying degrees of Alexa 680 conjugation toan anti-Jagged antibody 4D11 or anti-Jagged activatable antibody5342-1204-4D11 using thiol conjugatable Alexa 680 as a surrogate forthiol conjugatable toxin. This figure also demonstrates that suchconjugation can be effected so as to maintain activation of anti-Jaggedactivatable antibody by uPA.

FIG. 9A is a graph demonstrating the binding activities of anti-Jaggedantibody 4D11, antibody conjugate 4D11-vc-MMAD, activatable antibodyconjugate 5342-1204-4D11-vc-MMAD, uPA-activated activatable antibodyconjugate 5342-1204-4D11-vc-MMAD, and Synagis to the pancreaticadenocarcinoma cell line BxPC3. Cells were incubated with the respectivecompositions and then stained with AF-647-labelled anti human IgG.

FIG. 9B is a graph demonstrating cytotoxicity activities of anti-Jaggedantibody conjugate 4D11-vc-MMAD, activatable antibody conjugate5342-1204-4D1 I-vc-MMAD, uPA-activated activatable antibody conjugate5342-1204-4D1 I-vc-MMAD, and rituximab antibody conjugated to linkerpayload vc-MMAD on BxPC3 cells. Viability was measured using Cell TiterGlo reagent and relative Luminescence units plotted against dose.

FIG. 10A is a graph demonstrating the binding activities of anti-Jaggedantibody 4D11, antibody conjugate 4D11-vc-MMAE, activatable antibody5342-1204-4D11, activatable antibody conjugate 5342-1204-4D11-vc-MMAE,uPA-activated activatable antibody conjugate 5342-1204-4D11-vc-MMAE, andSynagis conjugated to linker payload vc-MMAE to the pancreaticadenocarcinoma cell line BxPC3. Cells were incubated with the respectivecompositions and then stained with AF-647-labelled anti human IgG.

FIG. 10B is a graph demonstrating cytotoxicity activities of anti-Jaggedantibody conjugate 4D11-vc-MMAE, activatable antibody conjugate5342-1204-4D11-vc-MMAE, uPA-activated activatable antibody conjugate5342-1204-4D11-vc-MMAE, and Synagis conjugated to linker payload vc-MMAEon BxPC3. Viability was measured using Cell Titer Glo reagent andrelative Luminescence units plotted against dose.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides conjugates that include an activatableantibody and methods of making these activatable antibody conjugates.Also provided are activatable antibodies having points of conjugationfor receiving a drug or label. The conjugates can be usedtherapeutically, diagnostically (e.g., in vitro or in vivo), for in vivoimaging, and for any of a variety of other therapeutic, diagnosticand/or prophylactic uses.

Generally, the compositions and methods provided herein include anactivatable antibody that includes an antibody or antibody fragment (AB)that specifically binds a target, where the AB is coupled to a maskingmoiety (MM) that decreases the ability of the AB to bind its target. Insome embodiments, the activatable antibody further includes a cleavablemoiety (CM) that is a substrate for a protease. The compositions andmethods provided herein enable the attachment of one or more agents toone or more cysteine residues in the AB without compromising theactivity (e.g., the masking, activating or binding activity) of theactivatable antibody. In some embodiments, the compositions and methodsprovided herein enable the attachment of one or more agents to one ormore cysteine residues in the AB without reducing or otherwisedisturbing one or more intrachain disulfide bonds within the activatableantibody. In some embodiments, the compositions and methods providedherein enable the attachment of one or more agents to one or morecysteine residues in the AB without reducing or otherwise disturbing oneor more disulfide bonds within the MM.

The compositions and methods provided herein produce an activatableantibody that is conjugated to one or more agents, e.g., any of avariety of therapeutic, diagnostic and/or prophylactic agents, withoutany of the agent(s) being conjugated to the MM of the activatableantibody. The compositions and methods provided herein produceconjugated activatable antibodies in which the MM retains the ability toeffectively mask the AB of the activatable antibody. In addition, such aconjugated activatable antibody retains the ability to be activated, andthe activated AB retains the ability to bind to its target.

In some embodiments, the compositions and methods provided herein do notcompromise the masking activity and/or masking efficiency of the MM inthe activatable antibody. For example, in situations where thecompositions and methods might be found to negatively impact the maskingactivity and/or masking efficiency of the MM in the activatableantibody, the compositions and methods decrease or otherwise disturb themasking activity and/or masking efficiency of the activatable antibodyby no more than 50%, no more than 40%, no more than 30%, no more than25%, no more than 200, no more than 15%, no more than 10%, no more than9%/0, no more than 8%, no more than 7%, no more than 6%, no more than5%, no more than 4%, no more than 3%, no more than 2% or no more than1%, as compared to the level of masking activity and/or maskingefficiency of the activatable antibody prior to conjugation or in theabsence of any conjugation.

In some embodiments, the compositions and methods provided herein do notcompromise the activating activity and/or activating efficiency of theactivatable antibody. For example, in situations where the compositionsand methods might be found to negatively impact the activating activityand/or activating efficiency, the compositions and methods decrease orotherwise disturb the activating activity and/or activating efficiencyof the activatable antibody by no more than 50%, no more than 40%, nomore than 30%, no more than 25%, no more than 20%, no more than 15%, nomore than 10%, no more than 9%, no more than 8%, no more than 7%, nomore than 6%, no more than 5%, no more than 4%, no more than 3%, no morethan 2% or no more than 1%, as compared to the level of activatingactivity and/or activating efficiency of the activatable antibody priorto conjugation or in the absence of any conjugation.

In some embodiments, the compositions and methods provided herein do notcompromise the binding activity of the activatable antibody. Forexample, in situations where the compositions and methods might be foundto negatively impact the binding activity, the compositions and methodsdecrease or otherwise disturb the binding activity of the activatableantibody by no more than 50%, no more than 40%, no more than 30%, nomore than 25%, no more than 20%, no more than 15%, no more than 10%, nomore than 9%, no more than 8%, no more than 7%, no more than 6%, no morethan 5%, no more than 4%, no more than 3%, no more than 2% or no morethan 1%, as compared to the level of binding activity of the activatableantibody prior to conjugation or in the absence of any conjugation.

The compositions and methods provided herein determine the combinationof reagents and reaction conditions to produce the desired partialreduction followed by conjugation. When reduction and subsequentconjugation is not controlled properly, activatable antibodies will becompletely reduced, and the masking efficiency of the activatableantibody is/will be compromised.

The conjugated activatable antibodies include an antibody orantigen-binding fragment thereof (AB) that specifically binds a target,and the AB is coupled to a masking moiety (MM), such that coupling ofthe MM to the AB decreases the ability of the antibody orantigen-binding fragment thereof to bind the target. In someembodiments, the MM is coupled to the AB via a cleavable moiety (CM)that includes a substrate for a protease, for example, a protease thatis co-localized with the target at a treatment site in a subject.Numerous studies have demonstrated the correlation of aberrant proteaselevels, e.g., uPA, legumain, MT-SP1, matrix metalloproteases (MMPs), insolid tumors. (See e.g., Murthy R V, et al. “Legumain expression inrelation to clinicopathologic and biological variables in colorectalcancer.” Clin Cancer Res. 11 (2005): 2293-2299; Nielsen B S, et al.“Urokinase plasminogen activator is localized in stromal cells in ductalbreast cancer.” Lab Invest 81 (2001): 1485-1501; Mook O R, et al. “Insitu localization of gelatinolytic activity in the extracellular matrixof metastases of colon cancer in rat liver using quenched fluorogenicDQ-gelatin.” J Histochem Cytochem. 51 (2003): 821-829).

The conjugated activatable antibodies provided herein include asubstrate for a protease, which is useful in leveraging the proteaseactivity in tumor cells for targeted conjugated antibody activation atthe site of treatment and/or diagnosis. The substrate selection processis used to identify substrates that have a number of desirablecharacteristics. For example, the selected substrates are systemicallystable (i.e., stable in the systemic circulation of a subject), aregenerally not susceptible to cleavage by circulating proteases such asplasmin, thrombin, tissue plasminogen activator (tPA) or a kallikrein(KLK) such as KLK-5 and/or KLK-7, are non-toxic, are generally notsusceptible to cleavage at potential sites of toxicity such as the skinby proteases such as ADAM 9, ADAM 10, ADAM 17 and/or kallikreins, suchas KLK-5 and KLK-7, and are active at an intended site of treatmentand/or diagnosis. In some embodiments, the identified substrates areselected for proteases that are dysregulated, due to, for example, beingoverexpressed or showing excess activity, or being less susceptible toprotease inhibition (due, e.g., to underexpression of the correspondinginhibitor or reduction in inhibitor activity) at an intended site oftherapy and/or diagnosis but are not typically expressed at or innormal, healthy or otherwise non-diseased or damaged tissue, and thenthe selected substrates are subsequently counter-screened againstproteases expressed in normal, e.g., non-diseased, tissue.

As a non-limiting example, the AB is a binding partner for any targetlisted in Table 1.

TABLE 1 Exemplary Targets 1-92-LFA-3 Alpha-4 integrin Alpha-V integrinalpha4beta1 integrin alpha4beta7 integrin AGR2 Anti-Lewis-Y Apelin Jreceptor APRIL B7-H4 BAFF BTLA C5 complement C-242 CD2 CD3 CD6 CD9 CD11aCD19 CD20 CD22 CD24 CD25 CD27 CD28 CD30 CD33 CD38 CD40 CD40L CD41 CD44CD47 CD51 CD52 CD56 CD64 CD70 CD74 CD80 CD81 CD86 CD95 CD117 CD125 CD132(IL-2RG) CD133 CD137 CD138 CD166 CD172A CD248 CEACAM5 (CEA) CEACAM6(NCA-90) CLAUDIN-3 CLAUDIN-4 cMet Collagen Cripto CSFR CSFR-1 CTLA-4CTGF CXCL10 CXCL13 CXCR1 CXCR2 CXCR4 CYR61 DL44 DLL4 DPP-4 EGFREndothelin B receptor (ETBR) EpCAM EPHA2 ERBB3 F protein of RSV FAPFGF-2 FGF8 FGFR1 FGFR2 FGFR3 FGFR4 Folate receptor G-CSF G-CSFR GD2 GITRGLUT1 GLUT4 GM-CSF GM-CSFR GP IIb/IIIa receptors Gp130 GPIIB/IIIA GPNMBGRP78 HER2/neu HGF hGH HVEM Hyaluronidase ICOS IFNalpha IFNbeta IFNgammaIgE IgE Receptor (FceRI) IGF IGF1R IL1B IL1R IL2 IL11 IL12 IL12p40IL-12R, IL-12Rbeta1 IL13 IL13R IL15 IL17 IL18 IL21 IL23 IL23R IL27/IL27R(wsx1) IL29 IL-31R IL31/IL31R IL2R IL4 IL4R IL6, IL6R Insulin ReceptorJagged Ligands Jagged 1 Jagged 2 LAG-3 LIF-R LIGHT MRP4 MUC1 Mucin-16Na/K ATPase Neutrophil elastase NGF Nicastrin Notch Receptors Notch 1Notch 2 Notch 3 Notch 4 NOV OSM-R OX-40 PAR2 PDGF-AA PDGF-BB PDGFRalphaPDGFRbeta PD-1 PD-L1 PD-L2 Phosphatidyl-serine P1GF PSCA PSMA RAAG12RAGE SLC44A4 Sphingosine 1 Phosphate STEAP1 TAPA1 TGFbeta TIGIT TIM-3TLR2 TLR4 TLR6 TLR7 TLR8 TLR9 TMEM31 TNFalpha TNFR TNFRS12A TRAIL-R1TRAIL-R2 Transferrin Transferrin receptor TRK-A TRK-B uPAR VAP1 VCAM-1VEGF VEGF-A VEGF-B VEGF-C VEGF-D VEGFR1 VEGFR2 VEGFR3 VISTA WISP-1WISP-2 WISP-3

As a nonlimiting example, the AB is or is derived from an antibodylisted in Table

TABLE 2 Exemplary sources for Abs Antibody Trade Name (antibody name)Target Avastin ™ (bevacizumab) VEGF Lucentis ™ (ranibizumab) VEGFErbitux ™ (cetuximab) EGFR Vectibix ™ (panitumumab) EGFR Remicade ™(infliximab) TNFα Humira ™ (adalimumab) TNFα Tysabri ™ (natalizumab)Integrinα4 Simulect ™ (basiliximab) IL2R Soliris ™ (eculizumab)Complement C5 Raptiva ™ (efalizumab) CD11a Bexxar ™ (tositumomab) CD20Zevalin ™ (ibritumomab tiuxetan) CD20 Rituxan ™ (rituximab) CD20Ocrelizumab CD20 Arzerra ™ (ofatumumab) CD20 Obinutuzumab CD20 Zenapax ™(daclizumab) CD25 Adcetris ™ (brentuximab vedotin) CD30 Myelotarg ™(gemtuzumab) CD33 Mylotarg ™ (gemtuzumab ozogamicin) CD33 Campath ™(alemtuzumab) CD52 ReoPro ™ (abiciximab) Glycoprotein receptor IIb/IIIaXolair ™ (omalizumab) IgE Herceptin ™ (trastuzumab) Her2 Kadcyla ™(trastuzumab emtansine) Her2 Synagis ™ (palivizumab) F protein of RSV(ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L (pertuzumab)Her2-neu (ertumaxomab) CD3/Her2-neu Orencia ™ (abatacept) CTLA-4(tanezumab) NGF (bavituximab) Phosphatidylserine (zalutumumab) EGFR(mapatumumab) EGFR (matuzumab) EGFR (nimotuzumab) EGFR ICR62 EGFR mAb528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (edrecolomab) EpCAM RAV12 RAAG12huJ591 PSMA Enbrel ™ (etanercept) TNF-R Amevive ™ (alefacept) 1-92-LFA-3Antril ™, Kineret ™ (ankinra) IL-1Ra GC1008 TGFbeta Notch, e.g., Notch 1Jagged 1 or Jagged 2 (adecatumumab) EpCAM (figitumumab) IGF1R(tocilizumab) IL-6 receptor Stelara ™ (ustekinumab) IL-12/IL-23 Prolia ™(denosumab) RANKL

In some embodiments, the AB binds Epidermal Growth Factor Receptor(EGFR). In some embodiments, the AB that binds EGFR includes one or moreof the heavy chain and/or light chain sequences shown below.

C225v5 Antibody Heavy Chain Nucleotide Sequence: (SEQ ID NO: 1)CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAACTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATGAC225v5 Antibody Heavy Chain Amino Acid Sequence (SEQ ID NO: 2)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEATHNHYTQKSLSLSPGC225v5 Antibody Light Chain Nucleotide Sequence: (SEQ ID NO: 10)CAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTAGC225v5 Antibody Light Chain Amino Acid Sequence: (SEQ ID NO: 16)QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* C225v4 Antibody Heavy Chain Nucleotide Sequence:(SEQ ID NO: 238)CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCAACGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAACTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATGAC225v4 Antibody Heavy Chain Amino Acid Sequence: (SEQ ID NO: 239)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPFPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K*C225v6 Antibody Heavy Chain Nucleotide Sequence: (SEQ ID NO: 240)CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAACTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAATGA]C225v6 Antibody Heavy Chain Amino Acid Sequence (SEQ ID NO: 241)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K*

In some embodiments, the AB binds interleukin 6 receptor (IL-6R). Insome embodiments, the AB that binds IL-6R includes one or more of theheavy chain and/or light chain sequences shown below.

Av1 Antibody Heavy Chain Amino Acid Sequence: (SEQ ID NO: 242)QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIGYISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAv1 Antibody Light Chain Amino Acid Sequence: (SEQ ID NO: 243)DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

In some embodiments, the AB binds a Jagged target, e.g., Jagged 1,Jagged 2 or both Jagged 1 and Jagged 2. In some embodiments, the AB thatbinds a Jagged target includes one or more of the heavy chain and/orlight chain sequences shown below.

4D11 Light Chain sequence: (SEQ ID NO: 244)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVYKSENRGEC4D11 Heavy Chain sequence: (SEQ ID NO: 245)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK4D11v2 Heavy Chain sequence (SEQ ID NO: 246)EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK4D11v2 Light Chain Sequence (SEQ ID NO: 247)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLXKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

In some embodiments, the AB that binds a Jagged target includes one ormore of the variable heavy chain and/or variable light chain sequencesshown below.

Variable Light Chain Amino Sequence Lc4 (SEQ ID NO: 248)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc4 (SEQ ID NO: 249)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFT1SRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc5 (SEQ ID NO: 250)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc5 (SEQ ID NO: 251)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PYHGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc7 (SEQ ID NO: 252)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc7 (SEQ ID NO: 253)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc8 (SEQ ID NO: 254)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc8 (SEQ ID NO: 255)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHI GRTNPFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc13 (SEQ ID NO: 256)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYLASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc13 (SEQ ID NO: 257)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc16 (SEQ ID NO: 258)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc16 (SEQ ID NO: 259)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PYYGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc19 (SEQ ID NO: 260)DIQMTOSPSSLSASVGDRVTITCRASQSISSYLNWYQ0KPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc19 (SEQ ID NO: 261)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQNGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc21 (SEQ ID NO: 262)DIQMIQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc21 (SEQ ID NO: 263)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc24 (SEQ ID NO: 264)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy ChaiN Amino Sequence Hc24 (SEQ ID NO: 265)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc26 (SEQ ID NO: 266)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc26 (SEQ ID NO: 267)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc27 (SEQ ID NO: 268)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ TKVEIKRVariable Heavy Chain Amino Sequence Hc27 (SEQ ID NO: 269)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFYGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc28 (SEQ ID NO: 270)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYLASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc28 (SEQ ID NO: 271)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc30 (SEQ ID NO: 272)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc30 (SEQ ID NO: 273)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAA AFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc31 (SEQ ID NO: 274)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc31 (SEQ ID NO: 275)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc32 (SEQ ID NO: 276)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc32 (SEQ ID NO: 277)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc37 (SEQ ID NO: 278)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc37 (SEQ ID NO: 279)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PHNGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc39 (SEQ ID NO: 280)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc39 (SEQ ID NO: 281)EVQLLESGGGLVQPGGSLRISCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc40 (SEQ ID NO: 282)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKRHeavy Chain Amino Sequence Hc40 (SEQ ID NO: 283)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP PFFGQFDYWGQGTLVTVSSVariable Light Chain Amino Sequence Lc47 (SEQ ID NO: 284)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKRVariable Heavy Chain Amino Sequence Hc47 (SEQ ID NO: 285)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGOGTLVTVSSVariable 4B2 Light Chain (SEQ ID NO: 286)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQ GTKVEIKRVariable 4B2 Heavy Chain (SEQ ID NO: 287)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable 4D11 Light Chain (SEQ ID NO: 288)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQ GTKVEIKRVariable 4D11 Heavy Chain (SEQ ID NO: 289)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable 4E7 Light Chain (SEQ ID NO: 290)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQ GTKVEIKRVariable 4E7 Heavy Chain (SEQ ID NO: 291)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable 4E11 Light Chain (SEQ ID NO: 292)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQ GTKVEIKRVariable 4E11 Heavy Chain (SEQ ID NO: 293)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEPMGQLTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI GGRSAFDYWGQGTLVTVSSVariable 6B7 Light Chain (SEQ ID NO: 294)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQALVAPLTFGQ GTKVEIKRVariable 6B7 Heavy Chain (SEQ ID NO: 295)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSSVariable 6F8 Light Chain (SEQ ID NO: 296)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQALVAPLTFGQ GTKVEIKRVariable 6F8 Heavy Chain (SEQ ID NO: 297)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSS

By way of non-limiting example, the CM includes an amino acid sequencethat is a substrate or is derived from a substrate that is cleaved byone or more of the following enzymes or proteases listed in Table 3.

TABLE 3 Exemplary Enzymes/Proteases ADAMS, ADAMTS, e.g. ADAM8 ADAM9ADAM10 ADAM12 ADAM15 ADAM17/TACE ADAMDEC1 ADAMTS1 ADAMTS4 ADAMTS5Aspartate proteases, e.g., BACE Renin Aspartic cathepsins, e.g.,Cathepsin D Cathepsin E Caspases, e.g., Caspase 1 Caspase 2 Caspase 3Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase 8 Caspase 9 Caspase 10Caspase 14 Cysteine cathepsins, e.g., Cathepsin B Cathepsin C CathepsinK Cathepsin L Cathepsin S Cathepsin V/L2 Cathepsin X/Z/P Cysteineproteinases, e.g., Cruzipain Legumain Otubain-2 KLKs, e.g., KLK4 KLK5KLK6 KLK7 KLK8 KLK10 KLK11 KLK13 KLK14 Metallo proteinases, e.g., MeprinNeprilysin PSMA BMP-1 MMPs, e.g., MMP1 MMP2 MMP3 MMP7 MMP8 MMP9 MMP10MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP20 MMP23 MMP24 MMP26MMP27 Serine proteases, e.g., activated protein C Cathepsin A CathepsinG Chymase coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa,FXIIa) Elastase Granzyme B Guanidinobenzoatase HtrA1 Human NeutrophilElastase Lactoferrin Marapsin NS3/4A PACE4 Plasmin PSA tPA ThrombinTryptase uPA Type II Transmembrane Serine Proteases (TTSPs), e.g., DESC1DPP-4 FAP Hepsin Matriptase-2 MT-SP1/Matriptase TMPRSS2 TMPRSS3 TMPRSS4

The conjugated activatable antibodies provided herein include a maskingmoiety. In some embodiments, the masking moiety is an amino acidsequence that is coupled or otherwise attached to the activatableantibody and is positioned within the activatable antibody constructsuch that the masking moiety decreases the ability of the antibody tospecifically bind the target. Suitable masking moieties are identifiedusing any of a variety of known techniques. For example, peptide maskingmoieties are identified using the methods described in U.S. Pat. No.8,293,685 by Daugherty et al., the contents of which are herebyincorporated by reference in their entirety.

In some embodiments, the masking moiety is selected for use with aspecific antibody or antibody fragment. For example, suitable maskingmoieties for use with antibodies that bind EGFR include MMs that includethe sequence CISPRG (SEQ ID NO: 29). By way of non-limiting examples,the MM can include a sequence such as CISPRGCG (SEQ ID NO: 30);CISPRGCPDGPYVMY (SEQ ID NO: 31); CISPRGCPDGPYVM (SEQ ID NO: 32),CISPRGCEPGTYVPT (SEQ ID NO: 33) and CISPRGCPGQIWHPP (SEQ ID NO: 34).Other suitable masking moieties include any of the EGFR-specific masksdisclosed in PCT Publication No. WO 2010/081173, such as, by way ofnon-limiting example, GSHCLIPINMGAPSC (SEQ ID NO: 35);CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 36); CNHHYFYTCGCISPRGCPG(SEQ ID NO: 37); ADHVFWGSYGCISPRGCPG (SEQ ID NO: 38);CHHVYWGHCGCISPRGCPG (SEQ ID NO: 39); CPHFTTTSCGCISPRGCPG (SEQ ID NO:40); CNHHYHYYCGCISPRGCPG (SEQ ID NO: 41); CPHVSFGSCGCISPRGCPG (SEQ IDNO: 42); CPYYTLSYCGCISPRGCPG (SEQ ID NO: 43); CNHVYFGTCGCISPRGCPG (SEQID NO: 44); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 45); CHHFTLTTCGCISPRGCPG(SEQ ID NO: 46); YNPCATPMCCISPRGCPG (SEQ ID NO: 47), CNHHYFYTCGCISPRGCG(SEQ ID NO: 48); CNHHYHYYCGCISPRGCG (SEQ ID NO: 49); CNHVYFGTCGCISPRGCG(SEQ ID NO: 50); CHHVYWGHCGCISPRGCG (SEQ ID NO: 51); CPHFTTTSCGCISPRGCG(SEQ ID NO: 52); CNHFTLTTCGCISPRGCG (SEQ ID NO: 53); CHHFTLTTCGCISPRGCG(SEQ ID NO: 54); CPYYTLSYCGCISPRGCG (SEQ ID NO: 55); CPHVSFGSCGCISPRGCG(SEQ ID NO: 56); ADHVFWGSYGCISPRGCG (SEQ ID NO: 57); YNPCATPMCCISPRGCG(SEQ ID NO: 58); CHHVYWGHCGCISPRGCG (SEQ ID NO: 59);C(N/P)H(H/V/F)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID NO:60); CISPRGCGQPIPSVK (SEQ ID NO: 61); CISPRGCTQPYHVSR (SEQ ID NO: 62);and/or CISPRGCNAVSGLGS (SEQ ID NO: 63).

Suitable masking moieties for use with antibodies that bind a Jaggedtarget, e.g., Jagged 1 and/or Jagged 2, include, by way of non-limitingexample, masking moieties that include a sequence such asQGQSGQCNIWLVGGDCRGWQG (SEQ ID NO: 232); QGQSGQGQQQWCNIWINGGDCRGWNG (SEQID NO: 64); PWCMQRQDFLRCPQP (SEQ ID NO: 65); QLGLPAYMCTFECLR (SEQ ID NO:66); CNLWVSGGDCGGLQG (SEQ ID NO: 67); SCSLWTSGSCLPHSP (SEQ ID NO: 68);YCLQLPHYMQAMCGR (SEQ ID NO: 69); CFLYSCTDVSYWNNT (SEQ ID NO: 70);PWCMQRQDYLRCPQP (SEQ ID NO: 71); CNLWISGGDCRGLAG (SEQ ID NO: 72);CNLWVSGGDCRGVQG (SEQ ID NO: 73); CNLWVSGGDCRGLRG (SEQ ID NO: 74);CNLWISGGDCRGLPG (SEQ ID NO: 75); CNLWVSGGDCRDAPW (SEQ ID NO: 76);CNLWVSGGDCRDLLG (SEQ ID NO: 77); CNLWVSGGDCRGLQG (SEQ ID NO: 78);CNLWLHGGDCRGWQG (SEQ ID NO: 79); CNIWLVGGDCRGWQG (SEQ ID NO: 80);CTTWFCGGDCGVMRG (SEQ ID NO: 81); CNIWGPSVDCGALLG (SEQ ID NO: 82);CNIWVNGGDCRSFEG (SEQ ID NO: 83); YCLNLPRYMQDMCWA (SEQ ID NO: 84);YCLALPHYMQADCAR (SEQ ID NO: 85); CFLYSCGDVSYWGSA (SEQ ID NO: 86);CYLYSCTDSAFWNNR (SEQ ID NO: 87); CYLYSCNDVSYWSNT (SEQ ID NO: 88);CFLYSCTDVSYW (SEQ ID NO: 89); CFLYSCTDVAYWNSA (SEQ ID NO: 90);CFLYSCTDVSYWGDT (SEQ ID NO: 91); CFLYSCTDVSYWGNS (SEQ ID NO: 92);CFLYSCTDVAYWNNT (SEQ ID NO: 93); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 94);CFLYSCTDVAYWSGL (SEQ ID NO: 95); CYLYSCTDGSYWNST (SEQ ID NO: 96);CFLYSCSDVSYWGNI (SEQ ID NO: 97); CFLYSCTDVAYW (SEQ ID NO: 98);CFLYSCTDVSYWGST (SEQ ID NO: 99); CFLYSCTDVAYWGDT (SEQ ID NO: 100);GCNIWLNGGDCRGWVDPLQG (SEQ ID NO: 101); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO:102); GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 103); GCNIWANGGDCRGWIDNIDG (SEQID NO: 104); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 105); GCNIWLVGGDCRGWLEEAVG(SEQ ID NO: 106); GGPALCNIWLNGGDCRGWSG (SEQ ID NO: 107);GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 108); GQQQWCNIWINGGDCRGWNG (SEQ ID NO:109); GKSEFCNIWLNGGDCRGWIG (SEQ ID NO: 110); GTPGGCNIWANGGDCRGWEG (SEQID NO: 111); GASQYCNLWINGGDCRGWRG (SEQ ID NO: 112); GCNIWLVGGDCRPWVEGG(SEQ ID NO: 113); GCNIWAVGGDCRPFVDGG (SEQ ID NO: 114);GCNIWLNGGDCRAWVDTG (SEQ ID NO: 115); GCNIWIVGGDCRPFINDG (SEQ ID NO:116); GCNIWLNGGDCRPVVFGG (SEQ ID NO: 117); GCNIWLSGGDCRMFMNEG (SEQ IDNO: 118); GCNIWVNGGDCRSFVYSG (SEQ ID NO: 119); GCNIWLNGGDCRGWEASG (SEQID NO: 120); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 121); GCNIWLNGGDCRTFVASG(SEQ ID NO: 122); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 123);GFLENCNIWLNGGDCRTG (SEQ ID NO: 124); GIYENCNIWLNGGDCRMG (SEQ ID NO:125); and/or GIPDNCNIWINGGDCRYG (SEQ ID NO: 126).

Suitable masking moieties for use with antibodies that bind aninterleukin 6 target, e.g., interleukin 6 receptor (IL-6R), include, byway of non-limiting example, masking moieties that include a sequencesuch as QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 127); QGQSGQGDFDIPFPAHWVPIT(SEQ ID NO: 128); QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 129);YRSCNWNYVSIFLDC (SEQ ID NO: 130); PGAFDIPFPAHWVPNT (SEQ ID NO: 131);ESSCVWNYVHIYMDC (SEQ ID NO: 132); YPGCKWNYDRIFLDC (SEQ ID NO: 133);YRTCSWNYVGIFLDC (SEQ ID NO: 134); YGSCSWNYVHIFMDC (SEQ ID NO: 135);YGSCSWNYVHIFLDC (SEQ ID NO: 136); YGSCNWNYVHIFLDC (SEQ ID NO: 137);YTSCNWNYVHIFMDC (SEQ ID NO: 138); YPGCKWNYDRIFLDC (SEQ ID NO: 139);WRSCNWNYAHIFLDC (SEQ ID NO: 140); WSNCHWNYVHIFLDC (SEQ ID NO: 141);DRSCTWNYVRISYDC (SEQ ID NO: 142); SGSCKWDYVHIFLDC (SEQ ID NO: 143);SRSCIWNYAHIHLDC (SEQ ID NO: 144); SMSCYWQYERIFLDC (SEQ ID NO: 145);YRSCNWNYVSIFLDC (SEQ ID NO: 146); YGSCSWNYVHIFMDC (SEQ ID NO: 147);SGSCKWDYVHIFLDC (SEQ ID NO: 148); YKSCHWDYVHIFLDC (SEQ ID NO: 149);YGSCTWNYVHIFMEC (SEQ ID NO: 150); FSSCNWNYVHIFLDC (SEQ ID NO: 151);WRSCNWNYAHIFLDC (SEQ ID NO: 152); YGSCQWNYVHIFLDC (SEQ ID NO: 153);YRSCNWNYVHIFLDC (SEQ ID NO: 154); NMSCHWDYVHIFLDC (SEQ ID NO: 155);FGPCTWNYARISWDC (SEQ ID NO: 156); XXsCXWXYvhlfXdC (SEQ ID NO: 157);MGVPAGCVWNYAHIFMDC (SEQ ID NO: 158); RDTGGQCRWDYVHIFMDC (SEQ ID NO:159); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 160); VGVPNGCVWNYAHIFMEC (SEQ IDNO: 161); DGGPAGCSWNYVHIFMEC (SEQ ID NO: 162); AVGPAGCWWNYVHIFMEC (SEQID NO: 163); CTWNYVHIFMDCGEGEGP (SEQ ID NO: 164); GGVPEGCTWNYAHIFMEC(SEQ ID NO: 165); AEVPAGCWWNYVHIFMEC (SEQ ID NO: 166);AGVPAGCTWNYVHIFMEC (SEQ ID NO: 167); SGASGGCKWNYVHIFMDC (SEQ ID NO:168); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 169); TPGCRWNYVHIFMECEAL (SEQ IDNO: 170); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 171); PGAFDIPFPAHWVPNT (SEQ IDNO: 172); RGACDIPFPAHWIPNT (SEQ ID NO: 173); QGDFDIPFPAHWVPIT (SEQ IDNO: 174); XGafDIPFPAHWvPnT (SEQ ID NO: 175); RGDGNDSDIPFPAHWVPRT (SEQ IDNO: 176); SGVGRDRDIPFPAHWVPRT (SEQ ID NO: 177); WAGGNDCDIPFPAHWIPNT (SEQID NO: 178); WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 179); AGSGNDSDIPFPAHWVPRT(SEQ ID NO: 180); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 181); and/orRECGRCGDIPFPAHWVPRT (SEQ ID NO: 182).

In some embodiments, the masking moiety is selected for use with anyantibody or antibody fragment. For example, in some embodiments, themasking moiety is a non-binding steric moiety (NB) or a binding partner(BP) for a non-binding steric moiety, where the BP recruits or otherwiseattracts the NB to the activatable antibody. For example, in someembodiments, the NB is a soluble, globular protein. In some embodiments,the NB is a protein that circulates in the bloodstream. In someembodiments, the NB is selected from the group consisting of albumin,fibrinogen, fibronectin, hemoglobin, transferrin, an immunoglobulindomain, and other serum proteins. In some embodiments, the BP isselected from the group consisting of an albumin binding peptide, afibrinogen binding peptide, a fibronectin binding peptide, a hemoglobinbinding peptide, a transferrin binding peptide, an immunoglobulin domainbinding peptide, and other serum protein binding peptides. In someembodiments, the activatable antibody has the structural arrangementfrom N-terminus to C-terminus as follows in the uncleaved state:NB-CM-AB, AB-CM-NB, BP-CM-AB or AB-CM-BP. In embodiments where theactivatable antibody includes a BP and the activatable antibody is inthe presence of the corresponding NB, the activatable antibody has astructural arrangement from N-terminus to C-terminus as follows in theuncleaved state: NB:BP-CM-AB or AB-CM-BP:NB, where “:” represents aninteraction, e.g., binding, between the NB and BP. In some embodiments,the activatable antibody has the structural arrangement from N-terminusto C-terminus as follows in the uncleaved state: NB-LP1-CM-LP2-AB,AB-LP2-CM-LP1-NB, BP-LP1-CM-LP2-AB or AB-LP2-CM-LP1-BP. In embodimentswhere the activatable antibody includes a BP and the activatableantibody is in the presence of the corresponding NB, the activatableantibody has a structural arrangement from N-terminus to C-terminus asfollows in the uncleaved state: NB:BP-LP1-CM-LP2-AB orAB-LP2-CM-LP1-BP:NB, where “:” represents an interaction, e.g., binding,between the NB and BP.

The conjugated activatable antibodies provided herein include acleavable moiety. In some embodiments, the cleavable moiety includes anamino acid sequence that is a substrate for a protease, usually anextracellular protease. Suitable substrates are identified using any ofa variety of known techniques. For example, peptide substrates areidentified using the methods described in U.S. Pat. No. 7,666,817 byDaugherty et al., the contents of which are hereby incorporated byreference in their entirety. (See also Boulware et al. “Evolutionaryoptimization of peptide substrates for proteases that exhibit rapidhydrolysis kinetics.” Biotechnol Bioeng. 106.3 (2010): 339-46).

In some embodiments, the CM is selected for use with a specificprotease. In some embodiments, the CM is a substrate for at least oneprotease selected from the group consisting of an ADAM 17, a BMP-1, acysteine protease such as a cathepsin, a HtrA1, a legumain, a matriptase(MT-SP1), a matrix metalloprotease (MMP), a neutrophil elastase, aTMPRSS, such as TMPRSS3 or TMPRSS4, a thrombin, and a u-type plasminogenactivator (uPA, also referred to as urokinase).

In some embodiments, the CM is a substrate for an ADAM 17. In someembodiments, the CM is a substrate for a BMP-1. In some embodiments, theCM is a substrate for a cathepsin. In some embodiments, the CM is asubstrate for a cysteine protease. In some embodiments, the CM is asubstrate for a HtrA1. In some embodiments, the CM is a substrate for alegumain. In some embodiments, the CM is a substrate for a MT-SP1. Insome embodiments, the CM is a substrate for a MMP. In some embodiments,the CM is a substrate for a neutrophil elastase. In some embodiments,the CM is a substrate for a thrombin. In some embodiments, the CM is asubstrate for a TMPRSS. In some embodiments, the CM is a substrate forTMPRSS3. In some embodiments, the CM is a substrate for TMPRSS4. In someembodiments, the CM is a substrate for uPA.

In some embodiments, the cleavable moiety is selected for use with aspecific protease, for example a protease that is known to beco-localized with the target of the activatable antibody. For example,suitable cleavable moieties for use in the activatable antibodies of thedisclosure include the sequence TGRGPSWV (SEQ ID NO: 183); SARGPSRW (SEQID NO: 184); TARGPSFK (SEQ ID NO: 185); LSGRSDNH (SEQ ID NO: 186);GGWHTGRN (SEQ ID NO: 187); HTGRSGAL (SEQ ID NO: 188); PLTGRSGG (SEQ IDNO: 189); AARGPAIH (SEQ ID NO: 190); RGPAFNPM (SEQ ID NO: 191); SSRGPAYL(SEQ ID NO: 192); RGPATPIM (SEQ ID NO: 193); RGPA (SEQ ID NO: 194);GGQPSGMWGW (SEQ ID NO: 195); FPRPLGITGL (SEQ ID NO: 196); VHMPLGFLGP(SEQ ID NO: 197); SPLTGRSG (SEQ ID NO: 198); SAGFSLPA (SEQ ID NO: 199);LAPLGLQRR (SEQ ID NO: 200); SGGPLGVR (SEQ ID NO: 201); and/or PLGL (SEQID NO: 202).

In some embodiments, the CM is a substrate for at least one matrixmetalloprotease (MMP). Examples of MMPs include MMP1; MMP2; MMP3; MMP7;MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17;MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. In some embodiments, theCM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP1, andMMP19. In some embodiments, the CM is a substrate for MMP7. In someembodiments the CM is a substrate for MMP9. In some embodiments, the CMis a substrate for MMP14. In some embodiments, the CM is a substrate fortwo or more MMPs. In some embodiments, the CM is a substrate for atleast MMP9 and MMP14. In some embodiments, the CM comprises two or moresubstrates for the same MMP. In some embodiments, the CM comprises atleast two or more MMP9 substrates. In some embodiments, the CM comprisesat least two or more MMP14 substrates.

In some embodiments, the CM is a substrate for an MMP and includes thesequence ISSGLLSS (SEQ ID NO: 298); QNQALRMA (SEQ ID NO: 299); AQNLLGMV(SEQ ID NO: 300); STFPFGMF (SEQ ID NO: 301); PVGYTSSL (SEQ ID NO: 302);DWLYWPGI (SEQ ID NO: 303); MIAPVAYR (SEQ ID NO: 304); RPSPMWAY (SEQ IDNO: 305); WATPRPMR (SEQ ID NO: 306); FRLLDWQW (SEQ ID NO: 307); LKAAPRWA(SEQ ID NO: 308); GPSHLVLT (SEQ ID NO: 309); LPGGLSPW (SEQ ID NO: 310);MGLFSEAG (SEQ ID NO: 311); SPLPLRVP (SEQ ID NO: 312); RMHLRSLG (SEQ IDNO: 313); LAAPLGLL (SEQ ID NO: 314); AVGLLAPP (SEQ ID NO: 315); LLAPSHRA(SEQ ID NO: 316); PAGLWLDP (SEQ ID NO: 317); and/or ISSGLSS (SEQ ID NO:318).

In some embodiments, activatable antibodies for use in the conjugatedactivatable antibodies of the disclosure may be made biosyntheticallyusing recombinant DNA technology and expression in eukaryotic orprokaryotic species. The cDNAs encoding the masking moiety, linkersequence (that may include a cleavable moiety (CM), and antibody chain(heavy or light)) can be linked in an 5′ to 3′ (N- to C-terminal in thetranslated product) sequence to create the nucleic acid construct, whichis expressed as the activatable antibody protein following aconventional antibody expression process. In some embodiments, theactivatable antibody could be semi-synthetically produced by expressinga CM-antibody and then coupling the mask chemically at or near theN-terminus of the protein. In some embodiments, the activatable antibodycould be produced by expressing an antibody and then coupling the maskand the CM chemically at or near the N-terminus of the protein such thatthe activatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM.

The conjugated activatable antibodies described herein also include anagent conjugated to the activatable antibody. In some embodiments, theconjugated agent is a therapeutic agent, such as an antineoplasticagent. In some embodiments, the agent is conjugated to a sulfhydrylgroup of the antibody or antigen-binding fragment in the activatableantibody. In some embodiments, the agent is a thiol-containing agent. Insome embodiments, the agent is engineered to include one or more thiolgroups.

In some embodiments, the agent is a cytotoxic agent such as a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). Suitable cytotoxic agents include, for example, any ofthe cytotoxic agents listed in Table 4.

In some embodiments, the agent is a thiol-containing agent. In someembodiments, the agent is engineered to include one or more thiolgroups. In some embodiments, the agent is a microtubule inhibitor. Insome embodiments, the agent is a dolastatin or a derivative thereof(e.g. auristatin E, AFP, MMAF, MMAE, MMAD, DMAF, DMAE). In someembodiments, the agent is monomethyl auristatin E (MMAE). In someembodiments, the agent is monomethyl auristatin D (MMAD). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a nucleic acid damaging agent. In some embodiments, the agentis a duocarmycin or derivative thereof. In some embodiments, the agentis a calicheamicin or derivative thereof.

In some embodiments, the agent is linked to the AB using a maleimidecaproyl-valine-citrulline linker or a maleimide PEG-valine-citrullinelinker. In some embodiments, the agent is linked to the AB using amaleimide caproyl-valine-citrulline linker. In some embodiments, theagent is linked to the AB using a maleimide PEG-valine-citrulline linkerIn some embodiments, the agent is monomethyl auristatin D (MMAD) linkedto the AB using a maleimidePEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and thislinker payload construct is referred to herein as “vc-MMAD.” In someembodiments, the agent is monomethyl auristatin E (MMAE) linked to theAB using a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyllinker, and this linker payload construct is referred to herein as“vc-MMAE.” The structures of vc-MMAD and vc-MMAE are shown below:

In some embodiments, in addition to the compositions and methodsprovided herein, the conjugated activatable antibody can also bemodified for site-specific conjugation through modified amino acidsequences inserted or otherwise included in the activatable antibodysequence. These modified amino acid sequences are designed to allow forcontrolled placement and/or dosage of the conjugated agent within aconjugated activatable antibody. For example, the activatable antibodycan be engineered to include cysteine substitutions at positions onlight and heavy chains that provide reactive thiol groups and do notnegatively impact protein folding and assembly, nor alter antigenbinding. In some embodiments, the activatable antibody can be engineeredto include or otherwise introduce one or more non-natural amino acidresidues within the activatable antibody to provide suitable sites forconjugation. In some embodiments, the activatable antibody can beengineered to include or otherwise introduce enzymatically activatablepeptide sequences within the activatable antibody sequence.

In some embodiments, the agent is a detectable moiety such as, forexample, a label or other marker. For example, the agent is or includesa radiolabeled amino acid, one or more biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods), one or more radioisotopes or radionuclides, oneor more fluorescent labels, one or more enzymatic labels, and/or one ormore chemiluminescent agents. In some embodiments, detectable moietiesare attached by spacer molecules.

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and agent are made using a variety ofbifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionuclide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Table 4 lists some of the exemplary pharmaceutical agents that may beemployed in the herein described invention but in no way is meant to bean exhaustive list.

TABLE 4 Exemplary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTSAuristatins Auristatin E Monomethyl auristatin D (MMAD) Monomethylauristatin E (MMAE) Desmethyl auristatin E (DMAE) Auristatin FMonomethyl auristatin F (MMAF) Desmethyl auristatin F (DMAF) Auristatinderivatives, e.g., amides thereof Auristatin tyramine Auristatinquinoline Dolastatins Dolastatin derivatives Dolastatin 16 DmJDolastatin 16 Dpv Maytansinoids, e.g. DM-1; DM-4 Maytansinoidderivatives Duocarmycin Duocarmycin derivatives Alpha-amanitinAnthracyclines Doxorubicin Daunorubicin Bryostatins CamptothecinCamptothecin derivatives 7-substituted Camptothecin10,11-Difluoromethylenedioxycamptothecin CombretastatinsDebromoaplysiatoxin Kahalalide-F Discodermolide EcteinascidinsANTIVIRALS Acyclovir Vira A Symmetrel ANTIFUNGALS Nystatin ADDITIONALANTI-NEOPLASTICS Adriamycin Cerubidine Bleomycin Alkeran Velban OncovinFluorouracil Methotrexate Thiotepa Bisantrene Novantrone ThioguanineProcarabizine Cytarabine ANTI-BACTERIALS Aminoglycosides StreptomycinNeomycin Kanamycin Amikacin Gentamicin Tobramycin Streptomycin BSpectinomycin Ampicillin Sulfanilamide Polymyxin ChloramphenicolTurbostatin Phenstatins Hydroxyphenstatin Spongistatin 5 Spongistatin 7Halistatin 1 Halistatin 2 Halistatin 3 Modified BryostatinsHalocomstatins Pyrrolobenzimidazoles (PBI) Cibrostatin6 DoxaliformAnthracyclins analogues Anthracyclins analogues Cemadotin analogue(CemCH2-SH) Pseudomonas toxin A (PE38) variant Pseudomonas toxin A(ZZ-PE38) variant ZJ-101 OSW-1 4-Nitrobenzyloxycarbonyl Derivatives ofO6-Benzylguanine Topoisomerase inhibitors Hemiasterlin CephalotaxineHomoharringtonine Pyrrolobenzodiazepine dimers (PBDs)Pyrrolobenzodiazepenes Functionalized pyrrolobenzodiazepenesCalicheamicins Podophyllotoxins Taxanes Vinca alkaloids CONJUGATABLEDETECTION REAGENTS Fluorescein and derivatives thereof Fluoresceinisothiocyanate (FITC) RADIOISOTOPES ¹²⁵I ¹³¹I ⁸⁹Zr ¹¹¹In ¹²³I ¹³¹I ⁹⁹mTc²⁰¹Tl ¹³³Xe ¹¹C ⁶²Cu ¹⁸F ⁶⁸Ga ¹³N ¹⁵O ³⁸K ⁸²Rb ⁹⁹mTc (Technetium) HEAVYMETALS Barium Gold Platinum ANTI-MYCOPLASMALS Tylosine Spectinomycin

In some embodiments, in addition to the compositions and methodsprovided herein, the conjugated activatable antibody can also be coupledusing any chemical reaction that will bind the two molecules so long asthe antibody and the other moiety retain their respective activities.This linkage can include many chemical mechanisms, for instance covalentbinding, affinity binding, intercalation, coordinate binding andcomplexation. In some embodiments, the binding is covalent binding.Covalent binding can be achieved either by direct condensation ofexisting side chains or by the incorporation of external bridgingmolecules. Many bivalent or polyvalent linking agents are useful incoupling protein molecules, such as the activatable antibodies of thepresent invention, to other molecules. For example, representativecoupling agents can include organic compounds such as thioesters,carbodiimides, succinimide esters, diisocyanates, glutaraldehyde,diazobenzenes and hexamethylene diamines. This listing is not intendedto be exhaustive of the various classes of coupling agents known in theart but, rather, is exemplary of the more common coupling agents. (SeeKillen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al.,Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science238:1098 (1987).

Suitable linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Suitablelinkers include: (i) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (ii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); and (iii) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G. Additional linkers include, but are not limited to, SMCC,sulfo-SMCC, SPDB, or sulfo-SPDB.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, the linker SMPT contains a sterically hindereddisulfide bond, and can form conjugates with increased stability.Disulfide linkages, are in general, less stable than other linkagesbecause the disulfide linkage is cleaved in vitro, resulting in lessconjugate available.

The reagent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride is useful to create a carboxamide starting with acarboxylic acid and a primary or secondary amine. Thus, EDC may be usedto link lysine residues in an antibody with a carboxylic acid in alinker or toxin, or to link aspartate or glutamate residues in anantibody with an amine in a linker or toxin. Such conjugation reactionsutilizing EDC may be enhanced by addition of NHS (N-hydroxysuccinimide)or sulfo-NHS (N-hydroxy-3-oxysulfonylsuccinimide). Addition of NHS orsulfo-NHS to such conjugation reactions may enhance the rate,completeness, selectivity, and/or reproducibility of the conjugationreactions.

In some embodiments, the linkers are cleavable. In some embodiments, thelinkers are non-cleavable. In some embodiments, two or more linkers arepresent. The two or more linkers are all the same, e.g., cleavable ornon-cleavable, or the two or more linkers are different, e.g., at leastone cleavable and at least one non-cleavable.

In some embodiments, in addition to the compositions and methodsprovided herein, the conjugated activatable antibody can also be furtherconjugated using any of several methods for attaching agents to ABs: (a)attachment to the carbohydrate moieties of the AB, or (b) attachment tosulfhydryl groups of the AB, or (c) attachment to amino groups of theAB, or (d) attachment to carboxylate groups of the AB. According to theinvention, ABs may be covalently attached to an agent through anintermediate linker having at least two reactive groups, one to reactwith AB and one to react with the agent. The linker, which may includeany compatible organic compound, can be chosen such that the reactionwith AB (or agent) does not adversely affect AB reactivity andselectivity. Furthermore, the attachment of linker to agent might notdestroy the activity of the agent. Suitable linkers for reaction withoxidized antibodies or oxidized antibody fragments include thosecontaining an amine selected from the group consisting of primary amine,secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine,semicarbazide and thiosemicarbazide groups. Such reactive functionalgroups may exist as part of the structure of the linker, or may beintroduced by suitable chemical modification of linkers not containingsuch groups.

According to the present invention, suitable linkers for attachment toreduced ABs include those having certain reactive groups capable ofreaction with a sulfhydryl group of a reduced antibody or fragment. Suchreactive groups include, but are not limited to: reactive haloalkylgroups (including, for example, haloacetyl groups), p-mercuribenzoategroups and groups capable of Michael-type addition reactions (including,for example, maleimides and groups of the type described by Mitra andLawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

According to the present invention, suitable linkers for attachment toneither oxidized nor reduced ABs include those having certain functionalgroups capable of reaction with the primary amino groups present inunmodified lysine residues in the AB. Such reactive groups include, butare not limited to, NHS carboxylic or carbonic esters, sulfo-NHScarboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonicesters, pentafluorophenyl carboxylic or carbonic esters, acylimidazoles, isocyanates, and isothiocyanates.

According to the present invention, suitable linkers for attachment toneither oxidized nor reduced ABs include those having certain functionalgroups capable of reaction with the carboxylic acid groups present inaspartate or glutamate residues in the AB, which have been activatedwith suitable reagents. Suitable activating reagents include EDC, withor without added NHS or sulfo-NHS, and other dehydrating agents utilizedfor carboxamide formation. In these instances, the functional groupspresent in the suitable linkers would include primary and secondaryamines, hydrazines, hydroxylamines, and hydrazides.

The agent may be attached to the linker before or after the linker isattached to the AB. In certain applications it may be desirable to firstproduce an AB-linker intermediate in which the linker is free of anassociated agent. Depending upon the particular application, a specificagent may then be covalently attached to the linker. In some embodimentsthe AB is first attached to the MM, CM and associated linkers and thenattached to the linker for conjugation purposes.

Branched Linkers:

In specific embodiments, branched linkers that have multiple sites forattachment of agents are utilized. For multiple site linkers, a singlecovalent attachment to an AB would result in an AB-linker intermediatecapable of binding an agent at a number of sites. The sites may bealdehyde or sulfhydryl groups or any chemical site to which agents canbe attached.

In some embodiments, higher specific activity (or higher ratio of agentsto AB) can be achieved by attachment of a single site linker at aplurality of sites on the AB. This plurality of sites may be introducedinto the AB by either of two methods. First, one may generate multiplealdehyde groups and/or sulfhydryl groups in the same AB. Second, one mayattach to an aldehyde or sulfhydryl of the AB a “branched linker” havingmultiple functional sites for subsequent attachment to linkers. Thefunctional sites of the branched linker or multiple site linker may bealdehyde or sulfhydryl groups, or may be any chemical site to whichlinkers may be attached. Still higher specific activities may beobtained by combining these two approaches, that is, attaching multiplesite linkers at several sites on the AB.

Cleavable Linkers:

Peptide linkers that are susceptible to cleavage by enzymes of thecomplement system, such as but not limited to urokinase, tissueplasminogen activator, trypsin, plasmin, or another enzyme havingproteolytic activity may be used in one embodiment of the presentinvention. According to one method of the present invention, an agent isattached via a linker susceptible to cleavage by complement. Theantibody is selected from a class that can activate complement. Theantibody-agent conjugate, thus, activates the complement cascade andreleases the agent at the target site. According to another method ofthe present invention, an agent is attached via a linker susceptible tocleavage by enzymes having a proteolytic activity such as a urokinase, atissue plasminogen activator, plasmin, or trypsin. These cleavablelinkers are useful in conjugated activatable antibodies that include anextracellular toxin, e.g., by way of non-limiting example, any of theextracellular toxins shown in Table 4.

Non-limiting examples of cleavable linker sequences are provided inTable 5.

TABLE 5 Exemplary Linker Sequences for Conjugation Amino AcidTypes of Cleavable Sequences Sequence Plasmin cleavable sequencesPro-urokinase PRFKIIGG (SEQ ID NO: 203) PRFRIIGG (SEQ ID NO: 204) TGFβSSRHRRALD (SEQ ID NO: 205) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 206)Staphylokinase SSSFDKGKYKKGDDA (SEQ ID NO: 207) SSSFDKGKYKRGDDA(SEQ ID NO: 208) Factor Xa cleavable sequences IEGR (SEQ ID NO: 209)IDGR (SEQ ID NO: 210) GGSIDGR (SEQ ID NO: 211) MMP cleavable sequencesGelatinase A PLGLWA (SEQ ID NO: 212) Collagenase cleavable sequencesCalf skin collagen  GPQGIAGQ (α1(I) chain) (SEQ ID NO: 213)Calf skin collagen  GPQGLLGA (α2(I) chain) (SEQ ID NO: 214)Bovine cartilage collagen  GIAGQ (α1(II) chain) (SEQ ID NO: 215)Human liver collagen  GPLGIAGI (α1(III) chain) (SEQ ID NO: 216)Human α₂M GPEGLRVG (SEQ ID NO: 217) Human PZP YGAGLGVV (SEQ ID NO: 218)AGLGVVER (SEQ ID NO: 219) AGLGISST (SEQ ID NO: 220) Rat α₁M EPQALAMS(SEQ ID NO: 221) QALAMSAI (SEQ ID NO: 222) Rat α₂M AAYHLVSQ(SEQ ID NO: 223) MDAFLESS (SEQ ID NO: 224) Rat α₁I₃(2J) ESLPVVAV(SEQ ID NO: 225) Rat α₁I₃(27J) SAPAVESE (SEQ ID NO: 226)Human fibroblast collagenase DVAQFVLT (SEQ ID NO: 227)(autolytic cleavages) VAQFVLTE (SEQ ID NO: 228) AQFVLTEG(SEQ ID NO: 229) PVQPIGPQ (SEQ ID NO: 230)

In addition, agents may be attached via disulfide bonds (for example,the disulfide bonds on a cysteine molecule) to the AB. Since many tumorsnaturally release high levels of glutathione (a reducing agent) this canreduce the disulfide bonds with subsequent release of the agent at thesite of delivery. In certain specific embodiments the reducing agentthat would modify a CM would also modify the linker of the conjugatedactivatable antibody.

Spacer Elements and Cleavable Elements:

In some embodiments, it may be necessary to construct the linker in sucha way as to optimize the spacing between the agent and the AB of theactivatable antibody. This may be accomplished by use of a linker of thegeneral structure:

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

In some embodiments, the linker may comprise a spacer element and acleavable element. The spacer element serves to position the cleavableelement away from the core of the AB such that the cleavable element ismore accessible to the enzyme responsible for cleavage. Certain of thebranched linkers described above may serve as spacer elements.

Throughout this discussion, it should be understood that the attachmentof linker to agent (or of spacer element to cleavable element, orcleavable element to agent) need not be effected by a particular mode ofattachment or reaction. Any reaction providing a product of suitablestability and biological compatibility is acceptable.

Serum Complement and Selection of Linkers:

According to one method of the present invention, when release of anagent is desired, an AB that is an antibody of a class that can activatecomplement is used. The resulting conjugate retains both the ability tobind antigen and activate the complement cascade. Thus, according tothis embodiment of the present invention, an agent is joined to one endof the cleavable linker or cleavable element and the other end of thelinker group is attached to a specific site on the AB. For example, ifthe agent has an hydroxy group or an amino group, it may be attached tothe carboxy terminus of a peptide, amino acid or other suitably chosenlinker via an ester or amide bond, respectively. For example, suchagents may be attached to the linker peptide via a carbodimide reaction.If the agent contains functional groups that would interfere withattachment to the linker, these interfering functional groups can beblocked before attachment and deblocked once the product conjugate orintermediate is made. The opposite or amino terminus of the linker isthen used either directly or after further modification for binding toan AB that is capable of activating complement.

Linkers (or spacer elements of linkers) may be of any desired length,one end of which can be covalently attached to specific sites on the ABof the activatable antibody. The other end of the linker or spacerelement may be attached to an amino acid or peptide linker.

Thus when these conjugates bind to antigen in the presence of complementthe amide or ester bond that attaches the agent to the linker will becleaved, resulting in release of the agent in its active form. Theseconjugates, when administered to a subject, will accomplish delivery andrelease of the agent at the target site, and are particularly effectivefor the in vivo delivery of pharmaceutical agents, antibiotics,antimetabolites, antiproliferative agents and the like as presented inbut not limited to those in Table 4.

Linkers for Release without Complement Activation:

In yet another application of targeted delivery, release of the agentwithout complement activation is desired since activation of thecomplement cascade will ultimately lyse the target cell. Hence, thisapproach is useful when delivery and release of the agent should beaccomplished without killing the target cell. Such is the goal whendelivery of cell mediators such as hormones, enzymes, corticosteroids,neurotransmitters, genes or enzymes to target cells is desired. Theseconjugates may be prepared by attaching the agent to an AB that is notcapable of activating complement via a linker that is mildly susceptibleto cleavage by serum proteases. When this conjugate is administered toan individual, antigen-antibody complexes will form quickly whereascleavage of the agent will occur slowly, thus resulting in release ofthe compound at the target site.

Biochemical Cross Linkers:

In some embodiments, the activatable antibody may be conjugated to oneor more therapeutic agents using certain biochemical cross-linkers.Cross-linking reagents form molecular bridges that tie togetherfunctional groups of two different molecules. To link two differentproteins in a step-wise manner, hetero-bifunctional cross-linkers can beused that eliminate unwanted homopolymer formation.

Peptidyl linkers cleavable by lysosomal proteases are also useful, forexample, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labilelinkers cleavable in the low-pH environment of the lysosome may be used,for example: bis-sialyl ether. Other suitable linkers includecathepsin-labile substrates, particularly those that show optimalfunction at an acidic pH.

Exemplary hetero-bifunctional cross-linkers are referenced in Table 6.

TABLE 6 Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONALCROSS-LINKERS Spacer Arm Length after Reactive Advantages andcross-linking Linker Toward Applications (Angstroms) SMPT Primary aminesGreater stability 11.2 Å Sulfhydryls SPDP Primary amines Thiolation  6.8Å Sulfhydryls Cleavable cross- linking LC-SPDP Primary amines Extendedspacer arm 15.6 Å Sulfhydryls Sulfo-LC- Primary amines Extender spacerarm 15.6 Å SPDP Sulfhydryls Water-soluble SMCC Primary amines Stablemaleimide 11.6 Å reactive group Sulfhydryls Enzyme-antibody conjugationHapten-carrier protein conjugation Sulfo-SMCC Primary amines Stablemaleimide 11.6 Å reactive group Sulfhydryls Water-solubleEnzyme-antibody conjugation MBS Primary amines Enzyme-antibody  9.9 Åconjugation Sulfhydryls Hapten-carrier protein conjugation Sulfo-MBSPrimary amines Water-soluble  9.9 Å Sulfhydryls SIAB Primary aminesEnzyme-antibody 10.6 Å conjugation Sulfhydryls Sulfo-SIAB Primary aminesWater-soluble 10.6 Å Sulfhydryls SMPB Primary amines Extended spacer arm14.5 Å Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary aminesExtended spacer arm 14.5 Å Sulfhydryls Water-soluble EDE/Sulfo- Primaryamines Hapten-Carrier 0 NHS conjugation Carboxyl groups ABHCarbohydrates Reacts with sugar 11.9 Å groups Nonselective

Non-Cleavable Linkers or Direct Attachment:

In some embodiments of the invention, the conjugate may be designed sothat the agent is delivered to the target but not released. This may beaccomplished by attaching an agent to an AB either directly or via anon-cleavable linker.

These non-cleavable linkers may include amino acids, peptides, D-aminoacids or other organic compounds that may be modified to includefunctional groups that can subsequently be utilized in attachment to ABsby the methods described herein. A-general formula for such an organiclinker could be

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

Non-Cleavable Conjugates:

In some embodiments, a compound may be attached to ABs that do notactivate complement. When using ABs that are incapable of complementactivation, this attachment may be accomplished using linkers that aresusceptible to cleavage by activated complement or using linkers thatare not susceptible to cleavage by activated complement.

Reducing Agents

Reducing Agent:

Examples of reducing agents suitable for use in the compositions andmethods of the disclosure include, by way of non-limiting example, BMS(bis(2-mercaptoethyl)sulfone), cysteamine, cysteine, DMH(dimethyl-bis-mercaptoacetyl hydrazine), DTBA (dithiobutylamine), DTT(dithiothreitol), GILT (gamma interferon inducible lysosomal thiolreductase; for enzymatic reduction), glutathione, β-mercaptoethanol, MEA(2-mercaptoethylamine), pyridine-2-thione, sodium borohydride, sodiumphosphorothioate, TCEP ((tris(2-carboxyethyl)phosphine)), andthiopropyl-agarose. In some embodiments, the reducing agent is DTT,β-mercaptoethanol or TCEP.

The studies provided herein use the reducing agent TCEP(tris(2-carboxyethyl)phosphine), which has the following structure:

TCEP is often used as a reducing agent to cleave disulfide bonds withinand between proteins. TCEP is very selective and does not react towardother functional groups found within proteins. TCEP does not react withburied disulfides.

Compared to the other two most common agents used for this purpose (DTTand β-mercaptoethanol), TCEP has the advantages of being odorless, amore powerful reducing agent, an irreversible reducing agent, morehydrophilic, and more resistant to oxidation in air. Unlike DTT, TCEP isactive at both alkaline and acidic conditions. TCEP is particularlyuseful when labeling cysteine residues with maleimides. TCEP can keepthe cysteines from forming disulfide bonds, and unlike DTT andf-mercaptoethanol, it will not react as readily with the maleimide.

The ratio of reduction agent to activatable antibody will vary dependingon the activatable antibody. In some embodiments, the ratio of reducingagent to activatable antibody will be in a range from about 20:1 to 1:1,from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1,from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1,from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to 1:1,from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1 to1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about 6:1to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, from about3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5, orfrom about 1:1 to 1:1.5. In some embodiments, the ratio is in a range offrom about 5:1 to 1:1. In some embodiments, the ratio is in a range offrom about 5:1 to 1.5:1. In some embodiments, the ratio is in a range offrom about 4:1 to 1:1. In some embodiments, the ratio is in a range fromabout 4:1 to 1.5:1. In some embodiments, the ratio is in a range fromabout 8:1 to about 1:1. In some embodiments, the ratio is in a range offrom about 2.5:1 to 1:1.

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab,and F(ab′)₂ fragments, scFvs, and an Fab expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≦1 μM; in some embodiments, the dissociation constant is ≦100 nM; insome embodiments, the dissociation constant is ≦10 nM.

As used herein, the terms “specific binding,” “immunological binding,”and “immunological binding properties” refer to the non-covalentinteractions of the type that occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(on)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofK_(off)/K_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present invention is said to specifically bind to atarget, when the dissociation binding constant (K_(d)) is ≦1 μM asmeasured by assays such as radioligand binding assays or similar assaysknown to those skilled in the art. In some embodiments, the K_(d) is≦100 nM. In some embodiments, the K_(d) is ≦10 nM. In some embodiments,the K_(d) is ≦1 nM. In some embodiments, the K_(d) is ≦100 μM to about 1μM.

The compositions and methods provided herein enable the attachment ofone or more agents to one or more cysteine residues in the AB withoutcompromising the activity (e.g., the masking, activating or bindingactivity) of the activatable antibody.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide that it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules shown herein, andnucleic acid molecules encoding the light chain immunoglobulin moleculesshown herein.

The term “isolated protein” referred to herein means a protein expressedfrom cDNA or recombinant RNA, or a protein of synthetic origin or somecombination thereof, which by virtue of its origin, or source ofderivation, the “isolated protein” (1) is not associated with proteinsfound in nature, (2) is free of other proteins from the same source, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules shown herein, and the light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andthat has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences that are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism: in prokaryotes andeukaryotes, such control sequences generally include promoter, ribosomalbinding site, and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. The term“polynucleotide” as referred to herein means nucleotides of at least 10bases in length, either ribonucleotides or deoxynucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. In some embodiments, oligonucleotides are10 to 60 bases in length. In some embodiments, the oligonucleotides are12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length.Oligonucleotides are usually single stranded, e.g., for probes, althougholigonucleotides may be double stranded, e.g., for use in theconstruction of a gene mutant. Oligonucleotides of the invention areeither sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotide linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, a εN-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction. Sequenceregions on the DNA strand having the same sequence as the RNA and thatare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”. Sequence regions on the DNA strand having the same sequenceas the RNA and that are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity. In some embodiments, the two peptide sequences shareat least 90 percent sequence identity. In some embodiments, the twopeptide sequences share at least 95 percent sequence identity. In someembodiments, the two peptide sequences share at least 99 percentsequence identity.

In some embodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75% amino acid sequenceidentity to a reference sequence (e.g., the wild-type sequence). In someembodiments, the variations in the amino acid sequence maintain at least80%, 90%, 95%, or 99% amino acid identity to the reference sequence. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids are generally divided into families: (1) acidic amino acidsare aspartate, glutamate; (2) basic amino acids are lysine, arginine,histidine; (3) non-polar amino acids are alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan, and (4)uncharged polar amino acids are glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. The hydrophilic amino acidsinclude arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) serine and threonine, which are thealiphatic-hydroxy family; (ii) asparagine and glutamine, which are theamide containing family; (iii) alanine, valine, leucine and isoleucine,which are the aliphatic family; and (iv) phenylalanine, tryptophan, andtyrosine, which are the aromatic family. For example, it is reasonableto expect that an isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid will not have a major effect on the binding or other properties ofthe resulting molecule, for example, in situations where the replacementdoes not involve an amino acid within a complementarity determiningregion (CDR) or other variable region. Whether an amino acid changeresults in a functional peptide can readily be determined by assayingthe specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. In some embodiments, amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. In some embodiments, computerized comparison methodsare used to identify sequence motifs or predicted protein conformationdomains that occur in other proteins of known structure and/or function.Methods to identify protein sequences that fold into a knownthree-dimensional structure are known. Bowie et al. Science 253:164(1991). Thus, the foregoing examples demonstrate that those of skill inthe art can recognize sequence motifs and structural conformations thatmay be used to define structural and functional domains in accordancewith the invention.

In some embodiments, amino acid substitutions are those that: (1)decrease susceptibility to proteolysis, (2) decrease susceptibility tooxidation, (3) alter binding affinity for forming protein complexes, (4)alter binding affinities, and (4) confer or modify other physicochemicalor functional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions (insome embodiments, conservative amino acid substitutions) may be made inthe naturally-occurring sequence (in some embodiments, in the portion ofthe polypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion and/or oneor more internal deletion(s), but where the remaining amino acidsequence is identical to the corresponding positions in thenaturally-occurring sequence deduced, for example, from a full lengthcDNA sequence. Fragments typically are at least 2, 3, 4, 5, 6, 7, 8, 9or 10 amino acids long. In some embodiments, the fragment is an antibodyfragment that is at least 14 amino acids long. In some embodiments, thefragment is a fragment of the AB that is least 20 amino acids long. Insome embodiments, the fragment is a fragment of the AB that is at least50 amino acids long. In some embodiments, the fragment is a fragment ofthe AB that is at least 70 amino acids long. The term “analog” as usedherein refers to polypeptides that are comprised of a segment of atleast 25 amino acids that has substantial identity to a portion of adeduced amino acid sequence and that has specific binding to a target,under suitable binding conditions. Typically, polypeptide analogscomprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, in some embodiments, atleast 50 amino acids long or longer, and can often be as long as afull-length naturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths todecrease potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

The term “drug” as used herein means an element, compound, agent, ormolecular entity, including, e.g., a pharmaceutical, therapeutic, orpharmacologic compound. Drugs can be natural or synthetic or acombination thereof. A “therapeutic drug” is an agent that exerts atherapeutic (e.g., beneficial) effect on cancer cells or immune cells(e.g., activated immune cells), either alone or in combination withanother agent (e.g., a prodrug converting enzyme in combination with aprodrug). Typically, therapeutic drugs useful in accordance with themethods and compositions described herein are those that exert acytotoxic, cytostatic, or immunosuppressive effect. In certainembodiments, a drug is not a radioactive element. The drug can be athiol-containing agent and/or the drug can be engineered to include oneor more thiol groups.

“Cytotoxic agent,” in reference to the effect of an agent on a cell,means killing of the cell. “Cytostatic agent” means an inhibition ofcell proliferation.

The term “interchain disulfide bond,” in the context of an antibody,refers to a disulfide bond between two heavy chains, or a heavy and alight chain.

The term “interchain thiol” refers to a thiol group of an antibody heavyor light chain that can participate in the formation of an interchaindisulfide bond.

A protein is referred to as “fully-loaded” when all points ofconjugation of a particular type and/or of similar reactivity areconjugated to drugs, resulting in a homogeneous population ofprotein-drug conjugate. A protein is referred to as “partially-loaded”when only some of the possible points of conjugation of a particulartype and/or of a similar reactivity are conjugated to drugs, resultingin formation of a certain isomer or isomers of the protein-drugconjugate.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and in someembodiments, a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, in some embodiments, more than about 85%, 90%, 95%, and99%. In some embodiments, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Use of Conjugated Activatable Antibodies

It will be appreciated that administration of conjugated activatableantibodies in accordance with the invention will be administered withsuitable carriers, excipients, and other agents that are incorporatedinto formulations to provide improved transfer, delivery, tolerance, andthe like. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2): 1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the invention, which include a conjugatedactivatable antibody, are used to prevent, treat or otherwise amelioratea disease or disorder associated with expression and/or activity of atarget. For example, therapeutic formulations of the invention are usedto treat or otherwise ameliorate a cancer or other neoplastic condition.In some embodiments the cancer is a solid tumor or a hematologicmalignancy where the target is expressed. In some embodiments the canceris a solid tumor where the target is expressed. In some embodiments thecancer is a hematologic malignancy where the target is expressed. Insome embodiments, the target is expressed on parenchyma (e.g., incancer, the portion of an organ or tissue that often carries outfunction(s) of the organ or tissue). In some embodiments, the target isexpressed on a cell, tissue, or organ. In some embodiments, the targetis expressed on stroma (i.e., the connective supportive framework of acell, tissue, or organ). In some embodiments, the target is expressed onan osteoblast. In some embodiments, the target is expressed on theendothelium (vasculature). In some embodiments, the target is expressedon a cancer stem cell. In some embodiments, the agent to which theactivatable antibody is conjugated is a microtubule inhibitor. In someembodiments, the agent to which the activatable antibody is conjugatedis a nucleic acid damaging agent.

Pathologies treated and/or prevented and/or for which the progression isdelayed and/or for which a symptom is ameliorated using the conjugatedactivatable anti-EGFR antibodies of the invention include, for example,diseases or disorders associated with expression and/or activity ofEGFR. In some embodiments, the disease or disorder associated withexpression and/or activity of EGFR is a cancer. In some embodiments, thecancer is a breast cancer, e.g., by way of non-limiting example, thebreast cancer is a triple-negative breast cancer. In some embodiments,the cancer is a triple-negative breast cancer. In some embodiments, thecancer is colorectal cancer. In some embodiments, the cancer is gastriccancer. In some embodiments, the cancer is glioblastoma. In someembodiments, the cancer is a head and neck cancer, e.g., by way ofnon-limiting example, esophageal cancer. In some embodiments, the canceris an esophageal cancer. In some embodiments, the cancer is a lungcancer, e.g., by way of non-limiting example, non-small cell lungcancer. In some embodiments, the cancer is a non-small cell lung cancer.In some embodiments, the cancer is ovarian/endometrial cancer. In someembodiments, the cancer is ovarian cancer. In some embodiments, thecancer is endometrial cancer. In some embodiments, the cancer ispancreatic cancer. In some embodiments, the cancer is prostate cancer.In some embodiments, the cancer is a renal cancer. In some embodiments,the cancer is a sarcoma, e.g., by way of non-limiting example,osteosarcoma. In some embodiments, the cancer is an osteosarcoma. Insome embodiments, the cancer is a skin cancer, e.g., by way ofnon-limiting example, squamous cell cancer, basal cell carcinoma, and/ormelanoma. In some embodiments, the cancer is a squamous cell cancer. Insome embodiments, the cancer is a skin squamous cell carcinoma. In someembodiments, the cancer is an esophageal squamous cell carcinoma. Insome embodiments, the cancer is a head and neck squamous cell carcinoma.In some embodiments, the cancer is a lung squamous cell carcinoma. Insome embodiments, the cancer is a basal cell carcinoma. In someembodiments, the cancer is a melanoma. In some embodiments, the agent towhich the activatable antibody is conjugated is a microtubule inhibitor.In some embodiments, the agent to which the activatable antibody isconjugated is a nucleic acid damaging agent.

In some embodiments, the indication, e.g., disease or disorderassociated with expression and/or activity of EGFR is an inflammatorydisorder and/or an autoimmune disease. In some embodiments, theinflammatory and/or autoimmune disease is psoriasis. In someembodiments, the agent to which the activatable antibody is conjugatedis a microtubule inhibitor. In some embodiments, the agent to which theactivatable antibody is conjugated is a nucleic acid damaging agent.

Pathologies treated and/or prevented and/or for which the progression isdelayed and/or for which a symptom is ameliorated using the conjugatedactivatable anti-Jagged antibodies of the invention include, forexample, cancer. In some embodiments, the conjugated activatableanti-Jagged antibodies of the invention are used to treat, prevent,delay the progression of, and/or ameliorate a symptom of a pathologysuch as, for example, leukemias, including T-cell acute lymphoblasticleukemia (T-ALL) and chronic lymphocytic leukemia (CLL), lymphoblasticdiseases including multiple myeloma, and solid tumors, including lung,colorectal, prostate, pancreatic and breast, including triple negativebreast cancer. In addition, since notch signaling is important for thesurvival and growth of cancer stem cells, inhibition of Jagged dependentnotch signaling would impact stem cell growth and survival.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, bonedisease or metastasis in cancer, regardless of primary tumor origin.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, breastcancer, including by way of non-limiting example, ER/PR+ breast cancer,Her2+ breast cancer, triple-negative breast cancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example,colorectal cancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, gastriccancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example,glioblastoma.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, headand neck cancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, lungcancer, such as by way of non-limiting example, non-small cell lungcancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example,multiple myeloma.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, ovariancancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example,pancreatic cancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example,prostate cancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example,sarcoma.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, renalcancer, such as by way of nonlimiting example, renal cell carcinoma.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, thyroidcancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, aurogenital cancer, such as bladder cancer, kidney cancer, or uterinecancer. In some embodiments, the pathology is bladder cancer. In someembodiments, the pathology is kidney cancer. In some embodiments, thepathology is uterine cancer.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, skincancer, such as by way of nonlimiting example, skin squamous cellcancer, such as esophageal squamous cell carcinoma (also known assquamous cell cancer of the esophagus), head and neck squamous cellcarcinoma (also known as squamous cell cancer of the head and neck) orlung squamous cell carcinoma (also known as squamous cell cancer of thelung), basal cell carcinoma, or melanoma. In some embodiments, thecancer is a squamous cell cancer. In some embodiments, the cancer is askin squamous cell carcinoma. In some embodiments, the cancer is anesophageal squamous cell carcinoma. In some embodiments, the cancer is ahead and neck squamous cell carcinoma. In some embodiments, the canceris a lung squamous cell carcinoma. In some embodiments, the cancer is abasal cell carcinoma. In some embodiments, the cancer is a melanoma.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chroniclymphoblastic leukemia (CLL) or myelodysplastic syndrome (MDS). In someembodiments, the pathology is ALL. In some embodiments, the pathology isAML. In some embodiments, the pathology is CLL. In some embodiments, thepathology is MDS.

In addition to cancer, Jagged-dependent notch signaling is critical toepithelial and fibroblast differentiation to myofibroblasts, cells witha central role in the development of fibrotic disease. Inhibition ofJagged dependent notch signaling, and therefore inhibition of theemergence of myofibroblasts, would be an effective treatment forfibrotic diseases of the kidney, liver, lung, and skin. In someembodiments, the conjugated activatable anti-Jagged antibodies are usedto treat a fibrotic disorder, such as idiopathic pulmonary fibrosis(IPF).

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example,fibrotic disease.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example,idiopathic pulmonary fibrosis, kidney fibrotic disease, liver fibroticdisease, peritoneal dialysis-induced fibrosis, scleroderma.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention are used to treat, prevent, delay the progression of,and/or ameliorate a symptom of a pathology such as, for example, hearingloss.

In some embodiments, the conjugated activatable anti-Jagged antibodiesof the invention that are used to treat, prevent, delay the progressionof, and/or ameliorate a symptom of such pathologies are conjugated to amicrotubule inhibitor agent. In some embodiments, the conjugatedactivatable anti-Jagged antibodies of the invention that are used totreat, prevent, delay the progression of, and/or ameliorate a symptom ofsuch pathologies are conjugated to a nucleic acid damaging agent.

Pathologies treated and/or prevented and/or for which the progression isdelayed and/or for which a symptom is ameliorated using the conjugatedactivatable anti-interleukin 6 receptor (IL-6) antibodies of theinvention include, for example, diseases or disorders associated withexpression and/or activity of IL-6R. In some embodiments, the disease ordisorder associated with expression and/or activity of IL-6R is cancer.In some embodiments, the cancer is breast cancer, including but notlimited to, triple negative breast cancer (TNBC). In some embodiments,the cancer is Castleman's disease. In some embodiments, the cancer ishepatocellular carcinoma. In some embodiments, the cancer is lungcancer. In some embodiments, the cancer is multiple myeloma. In someembodiments, the cancer is ovarian cancer. In some embodiments, thecancer is prostate cancer. In some embodiments, the agent to which theactivatable antibody is conjugated is a microtubule inhibitor. In someembodiments, the agent to which the activatable antibody is conjugatedis a nucleic acid damaging agent.

In some embodiments, the disease or disorder is inflammation and/or aninflammatory disorder. In some embodiments, the disease or disorder isan autoimmune disease. In some embodiments, the agent to which theactivatable antibody is conjugated is a microtubule inhibitor. In someembodiments, the agent to which the activatable antibody is conjugatedis a nucleic acid damaging agent.

Increased proteolysis is known to be a hallmark of cancer. (See e.g.,Affara N I, et al. “Delineating protease functions during cancerdevelopment.” Methods Mol Biol. 539 (2009): 1-32). Progression, invasionand metastasis of tumors result from several interdependent processes inwhich proteases are implicated.

Efficaciousness of prevention, amelioration or treatment is determinedin association with any known method for diagnosing or treating thedisease or disorder associated with target expression and/or activity.Prolonging the survival of a subject or otherwise delaying theprogression of the disease or disorder associated with target expressionand/or activity in a subject indicates that the activatable antibodyconfers a clinical benefit.

Conjugated activatable antibodies can be administered in the form ofpharmaceutical compositions. Principles and considerations involved inpreparing such compositions, as well as guidance in the choice ofcomponents are provided, for example, in Remington: The Science AndPractice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) MackPub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts,Possibilities, Limitations, And Trends, Harwood Academic Publishers,Langhome, Pa., 1994; and Peptide And Protein Drug Delivery (Advances InParenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

One embodiment of an activatable antibody fragment is the smallestfragment that specifically binds to the binding domain of the targetprotein. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. (See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Theformulation can also contain more than one active compound as necessaryfor the particular indication being treated, and in some embodiments,those with complementary activities that do not adversely affect eachother. Alternatively, or in addition, the composition can comprise anagent that enhances its function, such as, for example, a cytotoxicagent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, where matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

In some embodiments, the activatable antibody contains a detectablelabel. An intact antibody, or a fragment thereof (e.g., Fab, scFv, orF(ab)₂) is used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the invention can beused to detect a protein, polypeptide or peptide in a biological samplein vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte protein include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations, immunochemicalstaining, and immunofluorescence. Procedures for conducting immunoassaysare described, for example in “ELISA: Theory and Practice: Methods inMolecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa,N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, AcademicPress, Inc., San Diego, Calif., 1996; and “Practice and Theory of EnzymeImmunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detection of an analyte proteininclude introducing into a subject a labeled anti-analyte proteinantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

Diagnostic and Prophylactic Formulations

The conjugated activatable antibodies of the invention are used indiagnostic and prophylactic formulations. In one embodiment, aconjugated activatable antibody is administered to patients that are atrisk of developing one or more of the aforementioned cancer or otherdisorders. A patient's or organ's predisposition to one or more of theaforementioned disorders can be determined using genotypic, serologicalor biochemical markers.

In some embodiments of the invention, a conjugated activatable antibodyis administered to human individuals diagnosed with a clinicalindication associated with one or more of the aforementioned disorders.Upon diagnosis, a conjugated activatable antibody is administered tomitigate or reverse the effects of the clinical indication.

Conjugated activatable antibodies of the invention are also useful inthe detection of a target in patient samples and accordingly are usefulas diagnostics. For example, the conjugated activatable antibodies ofthe invention are used in in vitro assays, e.g., ELISA, to detect targetlevels in a patient sample.

Conjugated activatable antibodies can also be used in diagnostic and/orimaging methods. In some embodiments, such methods are in vitro methods.In some embodiments, such methods are in vivo methods. In someembodiments, such methods are in situ methods. In some embodiments, suchmethods are ex vivo methods. For example, a conjugated activatableantibody having an enzymatically cleavable CM can be used to detect thepresence or absence of an enzyme that is capable of cleaving the CM.Such conjugated activatable antibodies can be used in diagnostics, whichcan include in vivo detection (e.g., qualitative or quantitative) ofenzyme activity (or, in some embodiments, an environment of increasedreduction potential such as that which can provide for reduction of adisulfide bond) through measured accumulation of activated conjugatedactivatable antibodies (i.e., antibodies resulting from cleavage of aconjugated activatable antibody) in a given cell or tissue of a givenhost organism. Such accumulation of activated conjugated antibodiesindicates not only that the tissue expresses enzymatic activity (or anincreased reduction potential depending on the nature of the CM) butalso that the tissue expresses target to which the activated antibodybinds.

For example, the CM can be selected to be a protease substrate for aprotease found at the site of a tumor, at the site of a viral orbacterial infection at a biologically confined site (e.g., such as in anabscess, in an organ, and the like), and the like. The AB can be onethat binds a target antigen. Using methods as disclosed herein or, whenappropriate, methods familiar to one skilled in the art, a detectablelabel (e.g., a fluorescent label or radioactive label or radiotracer)can be conjugated to an AB or other region of an activatable antibody.Suitable detectable labels are discussed in the context of the abovescreening methods and additional specific examples are provided below.Using an AB specific to a protein or peptide of the disease state, alongwith a protease whose activity is elevated in the disease tissue ofinterest, protease-activated activatable antibodies will exhibit anincreased rate of binding to disease tissue relative to tissues wherethe CM specific enzyme is not present at a detectable level or ispresent at a lower level than in disease tissue or is inactive (e.g., inzymogen form or in complex with an inhibitor). Since small proteins andpeptides are rapidly cleared from the blood by the renal filtrationsystem, and because the enzyme specific for the CM is not present at adetectable level (or is present at lower levels in non-disease tissuesor is present in inactive conformation), accumulation of activatedantibodies in the disease tissue is enhanced relative to non-diseasetissues.

In another example, conjugated activatable antibodies can be used todetect the presence or absence of a cleaving agent in a sample. Forexample, where the conjugated activatable antibodies contain a CMsusceptible to cleavage by an enzyme, the conjugated activatableantibodies can be used to detect (either qualitatively orquantitatively) the presence of an enzyme in the sample. In anotherexample, where the conjugated activatable antibodies contain a CMsusceptible to cleavage by reducing agent, the conjugated activatableantibodies can be used to detect (either qualitatively orquantitatively) the presence of reducing conditions in a sample. Tofacilitate analysis in these methods, the conjugated activatableantibodies can be detectably labeled and can be bound to a support(e.g., a solid support, such as a slide or bead). The detectable labelcan be positioned on a portion of the activatable antibody that is notreleased following cleavage, for example, the detectable label can be aquenched fluorescent label or other label that is not detectable untilcleavage has occurred. The assay can be conducted by, for example,contacting the immobilized, detectably labeled activatable antibodieswith a sample suspected of containing an enzyme and/or reducing agentfor a time sufficient for cleavage to occur, then washing to removeexcess sample and contaminants. The presence or absence of the cleavingagent (e.g., enzyme or reducing agent) in the sample is then assessed bya change in detectable signal of the activatable antibodies prior tocontacting with the sample e.g., the presence of and/or an increase indetectable signal due to cleavage of the activatable antibody by thecleaving agent in the sample.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the AB ofthe conjugated activatable antibodies when cleaved. Thus, the assays canbe adapted to assess the presence or absence of a cleaving agent and thepresence or absence of a target of interest. The presence or absence ofthe cleaving agent can be detected by the presence of and/or an increasein detectable label of the activatable antibodies as described above,and the presence or absence of the target can be detected by detectionof a target-AB complex e.g., by use of a detectably labeled anti-targetantibody.

Conjugated activatable antibodies are also useful in in situ imaging forthe validation of activatable antibody activation, e.g., by proteasecleavage, and binding to a particular target. In situ imaging is atechnique that enables localization of proteolytic activity and targetin biological samples such as cell cultures or tissue sections. Usingthis technique, it is possible to confirm both binding to a given targetand proteolytic activity based on the presence of a detectable label(e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived froma disease site (e.g. tumor tissue) or healthy tissues. These techniquesare also useful with fresh cell or tissue samples.

In these techniques, an activatable antibody is labeled with adetectable label. The detectable label may be a fluorescent dye, (e.g.Fluor® Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), an AlexaFluor® label, such as Alexa Fluor® 680 or Alexa Fluor® 750), a nearinfrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, ahapten, a radioactive marker, biotin and an amplification reagent suchas streptavidin, or an enzyme (e.g., horseradish peroxidase or alkalinephosphatase).

Detection of the label in a sample that has been incubated with thelabeled, activatable antibody indicates that the sample contains thetarget and contains a protease that is specific for the CM of theactivatable antibody. In some embodiments, the presence of the proteasecan be confirmed using broad spectrum protease inhibitors and/or usingan agent that is specific for the protease, for example, an antibodysuch as A11, which is specific for the protease matriptase (MT-SP1) andinhibits the proteolytic activity of MT-SP1; see e.g., InternationalPublication Number WO 2010/129609, published 11 Nov. 2010. The sameapproach of using broad spectrum protease inhibitors and/or by using amore selective inhibitory agent can be used to identify a protease orclass of proteases specific for the CM of the activatable antibody. Insome embodiments, the presence of the target can be confirmed using anagent that is specific for the target, e.g., another antibody, or thedetectable label can be competed with unlabeled target. In someembodiments, unlabeled activatable antibody could be used, withdetection by a labeled secondary antibody or more complex detectionsystem.

Similar techniques are also useful for in vivo imaging where detectionof the fluorescent signal in a subject, e.g., a mammal, including ahuman, indicates that the disease site contains the target and containsa protease that is specific for the CM of the activatable antibody.

These techniques are also useful in kits and/or as reagents for thedetection, identification or characterization of protease activity in avariety of cells, tissues, and organisms based on the protease-specificCM in the activatable antibody.

In some embodiments, in situ imaging and/or in vivo imaging are usefulin methods to identify which patients to treat. For example, in in situimaging, the activatable antibodies are used to screen patient samplesto identify those patients having the appropriate protease(s) andtarget(s) at the appropriate location, e.g., at a tumor site.

In some embodiments in situ imaging is used to identify or otherwiserefine a patient population suitable for treatment with a conjugatedactivatable antibody of the disclosure. For example, patients that testpositive for both the target and a protease that cleaves the substratein the cleavable moiety (CM) of the activatable antibody being tested(e.g., accumulate activated antibodies at the disease site) areidentified as suitable candidates for treatment with such an activatableantibody comprising such a CM. Likewise, patients that test negative foreither or both of the target and the protease that cleaves the substratein the CM in the activatable antibody being tested using these methodsmight be identified as suitable candidates for another form of therapy.In some embodiments, such patients that test negative with respect to afirst activatable antibody can be tested with other activatableantibodies comprising different CMs until a suitable activatableantibody for treatment is identified (e.g., an activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).In some embodiments, the patient is then administered a therapeuticallyeffective amount of the conjugated activatable antibody for which thepatient tested positive.

In some embodiments in vivo imaging is used to identify or otherwiserefine a patient population suitable for treatment with an activatableantibody of the disclosure. For example, patients that test positive forboth the target and a protease that cleaves the substrate in thecleavable moiety (CM) of the activatable antibody being tested (e.g.,accumulate activated antibodies at the disease site) are identified assuitable candidates for treatment with such an activatable antibodycomprising such a CM. Likewise, patients that test negative might beidentified as suitable candidates for another form of therapy. In someembodiments, such patients that test negative with respect to a firstactivatable antibody can be tested with other activatable antibodiescomprising different CMs until a suitable activatable antibody fortreatment is identified (e.g., an activatable antibody comprising a CMthat is cleaved by the patient at the site of disease). In someembodiments, the patient is then administered a therapeuticallyeffective amount of the conjugated activatable antibody for which thepatient tested positive.

Pharmaceutical Compositions

The conjugated activatable antibodies of the invention (also referred toherein as “active compounds”), and derivatives, fragments, analogs andhomologs thereof, can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise theconjugated activatable antibody and a pharmaceutically acceptablecarrier. As used herein, the term “pharmaceutically acceptable carrier”is intended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Examples of suchcarriers or diluents include, but are not limited to, water, saline,ringer's solutions, dextrose solution, and 5% human serum albumin.Liposomes and non-aqueous vehicles such as fixed oils may also be used.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be suitable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Materials and Methods

The examples provided herein use an anti-EGFR activatable antibodyreferred to herein as activatable antibody 3954-1204-C225v5 (alsoreferred to herein as 3954-1204-C225v5 activatable antibody or3954-1204-C225v5) that includes an EGFR-binding sequence, a maskingmoiety (MM), and a cleavable moiety (CM) that is a substrate for aprotease. These examples also use an activatable anti-EGFR antibodyconstruct referred to herein as masked antibody 3954-NSUB-C225v5 (alsoreferred to herein as 3954-NSUB-C225v5 masked antibody or3954-NSUB-C225v5) that includes a non-cleavable moiety located betweenthe MM and the EGFR-binding sequence. It is to be understood that whilethe examples provided herein use these anti-EGFR activatable antibodyconstructs, these methods are applicable to any activatable antibodyhaving two or more cysteine residues, where it is desired that only aportion of the total number of cysteine residues in the activatableantibody be reduced prior to conjugation. This is referred to herein as“partial reduction.”

It should be further understood that the examples provided herein use afluorescent agent, Alexa-680 Fluor® (also referred to herein as Alexa680®), as the agent that is to be conjugated to an activatable antibody.This particular dye was chosen because it has a molecular weight that issimilar to a known cytotoxic agent, MMAE. However, this fluorescentagent is merely used as an example, and the compositions and methodsused herein are useful with any number of conjugated agents, includingby way of non-limiting example, toxins and other payload agents. Thecompositions and methods are not limited to agents of any particularmolecular weight, size or other such characteristic.

Anti-EGFR Activatable Antibody Constructs:

The 3954-1204-C225v5 activatable anti-EGFR antibody construct includesthe following heavy and light chain sequences:

3954-1204-C225v5 Activatable Antibody Heavy Chain Nucleotide Sequence:[C225v5 (SEQ ID NO: 1)] (SEQ ID NO: 1)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatg a]3954-1204-C225v5 Activatable Antibody Heavy Chain Amino Acid Sequence:[C225v5 (SEQ ID NO: 2)] (SEQ ID NO: 2)[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFHWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K*]3954-1204-C225v5 Activatable Antibody Light Chain Nucleotide Sequence:[Spacer (SEQ ID NO: 5)][Mask (SEQ ID NO: 6)] [ Linker 1 (SEQ ID NO: 7)][ 1204 Substrate (SEQ ID NO: 8)][

 (SEQ ID NO: 9)] [C225 (SEQ ID NO: 10)] (SEQ ID NO: 3)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac][ggctcgagcggtggcagcggtggctctggtggatccggt][ctgagcggccgttccgataatcat ] [

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacag gggagagtgttag] Bold:Spacer Underline: Mask Italics and Underline : Linker 1Bold and Underline : 1204 Substrate

: Linker Normal text: anti-EGFR  antibody de- rived sequence3954-1204-C225v5 Activatable Antibody Light Chain Amino Acid Sequence:[Spacer (SEQ ID NO: 11)][Mask (SEQ ID NO: 12)] [ Linker 1 (SEQ ID NO: 13)][ 1204 Substrate (SEQ ID NO: 14)][

 (SEQ ID NO: 15)] [C225 (SEQ ID NO: 16)] (SEQ ID NO: 4)[QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][ LSGRSDNH ] [

][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*] Bold: Spacer Underline: MaskItalics and Underline : Linker 1 Bold and Underline : 1204 Substrate

: Linker 2 Normal text: anti-EGFR   antibody de- rived sequence

In some embodiments, the spacer sequence for the light chain3954-1204-C225v5 activatable antibody can include an N-terminal variant,such as for example, a spacer selected from the group consisting ofGQSGQ (SEQ ID NO: 235), QSGQ (SEQ ID NO: 236), SGQ (SEQ ID NO: 237), GQand Q. In these embodiments, all other elements of the 3954-1204-C225v5activatable antibodies, e.g., the heavy chain sequence, the light chainsequence, the 3954 mask, linker 1, the 1204 substrate, and linker 2, allremain the same as shown above in SEQ ID NO: 4.

The 3954-NSUB-C225v5 masked anti-EGFR antibody construct includes thesame heavy chain as the 3954-1204-C225v5 activatable anti-EGFR antibodyshown above. The 3954-NSUB-C225v5 masked anti-EGFR antibody constructincludes the following light chain sequence:

3954-NSUB-C225v5 Masked Antibody Light Chain Nucleotide Sequence:[Spacer (SEQ ID NO: 5)][Mask (SEQ ID NO: 6)] [Linker 1-Noncleavable Substrate-Linker 2(SEQ ID NO: 19)][C225 (SEQ ID NO: 10)] (SEQ ID NO: 17)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacgg cccatacgtcatgtac][ggctcgagcggtggcagcggtggctctggtgg ctcaggtggaggctcgggcggtgggagcggcggttct][cagatcttgct gacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: MaskItalics and Underline : Linker 1-Noncleavable substrate-Linker 2Normal text: anti-EGFR antibody derived sequence3954-NSUB-C225v5 Masked Antibody Light Chain Amino Acid Sequence:[Spacer (SEQ ID NO: 11)][Mask (SEQ ID NO: 12)] [Linker 1-Noncleavable Substrate-Linker 2(SEQ ID NO: 20)][C225 (SEQ ID NO: 16)] (SEQ ID NO: 18)[QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSGGGSGGGSGGS ][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC*]Bold: Spacer Underline: Mask Italics and Underline :Linker 1-Noncleavable substrate-Linker 2 Normal text:anti-EGFR antibody derived sequence

Reducing Agent:

The studies provided herein use the reducing agent TCEP(tris(2-carboxyethyl)phosphine).

Protocol for TCEP Partial Reduction of Anti-EGFR Activatable Antibodyand Subsequent Conjugation to Maleimide Alexa-680:

Bond-Breaker@ TCEP Solution (neutral pH solution, Thermo Scientific) isused at various molar ratios of TCEP to an activatable antibody that, inthe cleaved state (i.e., activated state), binds Epidermal Growth FactorReceptor, and the anti-EGFR activatable antibody is formulated in PBS.For example, the ratio of reducing agent, e.g., TCEP, to activatableantibody to be tested can include a ratio in a range from about 20:1 to1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, fromabout 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5,or from about 1:1 to 1:1.5. In some embodiments, the ratio is in a rangeof from about 5:1 to 1:1. In some embodiments, the ratio is in a rangeof from about 5:1 to 1.5:1. In some embodiments, the ratio is in a rangeof from about 4:1 to 1:1. In some embodiments, the ratio is in a rangefrom about 4:1 to 1.5:1. In some embodiments, the ratio is in a rangefrom about 8:1 to about 1:1. In some embodiments, the ratio is in arange of from about 2.5:1 to 1:1. It is to be understood that while theexamples provided herein use an anti-EGFR activatable antibody referredto herein as 3954-1204-C225v5, these methods are applicable to anyactivatable antibody having two or more cysteine residues, where it isdesired that only a portion of the total number of cysteine residues inthe activatable antibody be reduced prior to conjugation. This isreferred to herein as “partial reduction.”

Briefly, a TCEP solution at twice the final concentration was mixed 1:1(volume:volume) with 3954-1204-C225v5 to result in the finalTCEP:(3954-1204-C225v5) ratio desired. The final solution was thenincubated at 37° C. for specified periods of time for the reductionreaction to progress. At the end of the reduction reaction, the solutionwas cooled to room temperature and Maleimide Alexa-680 (Invitrogen) wasadded into the solution (Maleimide Alexa-680 was used at half of thereduction volume and at a concentration equal to 10× molar concentrationof TCEP during the reduction reaction; for example, if the originalreduction reaction comprised 50 microliters (ul) of 13.2 uM3954-1204-C225v5 and 50 ul of 52.8 uM TCEP, then 50 ul 264 uM MaleimideAlexa-680 would be used) to begin the Alexa-680 conjugation. Theconjugation reaction proceeded for 2 hours at room temperature in alight tight container. After the 2-hour reaction, the solution was spundown and buffer exchanged into PBS using a PD-10 column (GE Healthcare)or equivalent using manufacturer's instructions. The final conjugatedproduct was analyzed using a UV Spectrophotometer to determine finalprotein concentration and the degree of labeling of the Alexa-680 dye.

Protocol for the Analysis of Maleimide Alexa-680 Conjugated Anti-EGFRActivatable Antibody Using LabChip GXII:

A HT Protein Express LabChip (Perkin Elmer) was prepared according tomanufacturer's instructions using either the Pico Protein Expressprotocol or the HT Protein Express protocol: the Pico Protein Expressprotocol was used to analyze the Alexa-680 conjugated portion of theTCEP reduced 3954-1204-C225v5; the HT Protein Express protocol was usedto analyze the total protein in the TCEP reduced, Alexa-680 conjugated3954-1204-C225v5. TCEP reduced, Alexa-680 conjugated 3954-1204-C225v5was prepared for the GXII analysis using Perkin Elmer's instructions.The sample was analyzed using the 200 series of the LabChip GXIIanalysis protocol (High sensitivity for the HT protocol and Pico for thePico protocol). Resulting data was analyzed using the LabChip GXIIsoftware.

Protocol for EGFR Binding ELISA:

NUNC Maxisorp flatbottom 96 well plates were coated with 50 ul/well, 2ug/ml human EGFR-Fc fusion protein (R&D Systems) in Hank's Balanced SaltSolution (HBSS, Teknova) for 2 hours at room temperature. At the end ofthe 2 hour coating, the liquid contents of the plate were evacuated and250 ul/well of HBSS containing 1% BSA was introduced and allowed toblock the plate for 30 minutes at room temperature. At the end of theblocking period, liquid contents of the 96-well plate were removed andserially diluted samples (i.e., 3954-1204-C225v5, Alexa-680 conjugated3954-1204-C225v5, uPA-activated 3954-1204-C225v5, uPA-activatedAlexa-680 conjugated 3954-1204-C225v5, and C225 (a cetuximab antibody),starting at a concentration of 100 ug/ml and diluted by a factor of 3per dilution step) were introduced at 50 ul/well. The plate wasincubated at room temperature for 1 hour. At the end of the hour, theplate was washed with HBSS containing 0.05% Tween-20 using a BioTekELx450 Select CW plate washer (300 ul/well wash volume, 6 cycles ofaspiration and wash). Washed plates were tapped dry and 50 ul/well of400 ng/ml Horse Radish Peroxidase conjugated Goat anti-Human IgG Fab′₂specific antibody (Jackson ImmunoResearch) were introduced and incubatedfor 30 minutes at room temperature. The plates were washed as previouslystated and 100 ul/well of 1-Step TMB Substrate (Thermo Scientific) wasintroduced. Color change was observed and the reaction was stopped bythe addition of 100 ul/well of 1M HCl (Fisher Scientific). The reactedplate was analyzed using a BioTek EL800 plate reader at O.D. 450. Datawere computed using Excel (Microsoft) and the result was plotted usingPrism 6 (GraphPad).

Example 2 TCEP-Mediated Reduction of Activatable Antibodies

The compositions and methods provided herein determine the combinationof reagents and reaction conditions that produce the desired partialreduction followed by conjugation. When reduction and subsequentconjugation is not controlled properly, activatable antibodies will becompletely reduced, and the masking efficiency of the activatableantibody is compromised. For example, when the reducing agent is used ata ratio of 20:1 (reducing agent to activatable antibody), theactivatable antibody was completely reduced into free heavy chain andfree light chain. Attempts to produce a milder reduction (i.e., lessthan complete reduction), for example, by immobilizing a reducing agent,were too mild and did not sufficiently reduce the activatable antibodyto allow for subsequent conjugation. In these studies, bandscorresponding to predominantly intact IgG (high molecular weightband >150 kDa) were observed in all reduction conditions.

Studies were conducted to determine the range of reducing agent toactivatable antibody. At lower ratios, for example, in the range of0.5:1 to 2:1 (reducing agent to activatable antibody), some reductionwas achieved, and the activatable antibody integrity and maskingefficiency were retained. At ratios of 1.5:1 to 5:1 (reducing agent toactivatable antibody), reduction time from 30 minutes to 2 hours, therewas an increasing amount of reduced activatable antibody speciescorresponding to the molecular weight of one heavy chain and one lightchain activatable antibody. The partially reduced activatable antibodymaintained the EGFR binding characteristics of the original non-reducedand masked activatable antibody demonstrating that the activatableantibody partially reduced under these conditions was capable ofmaintaining the original masking efficiency. At the identified ratio ofreducing agent to activatable antibody and reduction time, aninter-chain disulfide-reduced activatable antibody can be produced toallow for subsequent maximum conjugation through free cysteines whilemaintaining the masking efficiency of the original, non-reducedactivatable antibody.

At a reduction time of 2 hours, a ratio of reducing agent to activatableantibody ratio above 5:1 was too reductive to maintain the originalmasking efficiency of the tested activatable antibodies. The variedshift in masking efficiency loss and the varied amounts of partiallyreduced activatable antibody subspecies showed that the testedactivatable antibodies have different tolerance to reducingagent-mediated reduction, for example, TCEP-mediated reduction. Thevaried combination of antibody, linkers, cleavable moiety (CM) andmasking moiety (MM) results in a spectrum in the tolerance of theactivatable antibody for reducing agent-mediated reduction, for example,TCEP-mediated reduction.

In one set of studies described herein, an activatable anti-EGFRantibody referred to as 3954-1204-C225v5 was reduced at various ratiosof TCEP to activatable antibody (e.g., from about 1.5:1 to about 4:1)using a 90-minute reduction time. In some instances, reduction wasfollowed by conjugation to a fluorescent dye, Alexa 680. The results ofthese studies (at TCEP to activatable antibody ratios of 1.5:1, 2:1, and4:1) are shown in FIG. 1.

In another set of studies described herein, an anti-EGFR antibodyconstruct that includes an antigen-binding portion that specificallybinds EGFR, a masking moiety, and a non-cleavable linker (referred to as3954-NSUB-C225v5) was reduced at various ratios of TCEP to activatableantibody (e.g., from about 1.5:1 to about 4:1) using a 90-minutereduction time. In some instances, reduction was followed by conjugationto Alexa 680. The results of these studies (at TCEP to activatableantibody ratios of 1.5:1, 2:1, and 4:1) are shown in FIG. 2.

Using thiol conjugatable Alexa 680 as a surrogate for thiol conjugatabletoxin, these studies demonstrate varying degrees of Alexa 680conjugation dependent on both TCEP to activatable antibody ratio andtime of reduction. The conjugation of thiol conjugatable Alexa 680 toTCEP-partially reduced 3954-1204-C225v5 or 3954-NSUB-C225v5 does notsignificantly change the titration profile of 3954-1204-C225v5 or3954-NSUB-C225v5 to EGFR. Thus, partial reduction and subsequent thiolconjugation of Alexa 680 can be done in such a way as to maintain themasking efficiency of activatable antibodies. FIGS. 3 through 6demonstrate that partial reduction and subsequent thiol conjugation ofAlexa 680 Fluor® can be done in such a way as to also maintainactivation of 3954-1204-C225v5 by uPA and not to lead to activation of3954-NSUB-C225v5.

Further studies indicated that a degree of labeling (molar ratio ofAlexa 680 Fluor® vs. activatable antibody) of 3.8 was achieved for3954-1204-C225v5 and a degree of labeling of 3.5 was achieved for3954-NSUB-C225v5.

Example 3 Mass Spectrometry Analysis of Conjugated ActivatableAntibodies

Molecular Weight Determination by MALDI Mass Spectrometry (MALDI MS):

In MALDI MS, the dissolved sample is deposited on a metal target and thepeptides and proteins are co-crystallized with a light-absorbing matrix.A laser beam is directed at the dry matrix sample, the sample moleculesare desorbed and ionized and the masses are measured in a time-of-flight(TOF) mass analyzer. Proteins are observed in the mass spectrum(mass-over-charge spectrum m/z) as singly (m/z MH+) as well as multiplecharged ions.

In the present analysis, the partially reduced and conjugatedactivatable antibodies (intact and DTT-reduced) were purified using C4ziptips from Millipore. Each purified sample was mixed with2,5-dihydroxyacetophonone/diammonium hydrogen citrate (DHAP/DAHC) matrixand spotted onto a Big Anchor target from Bruker. Mass spectra wereobtained on an Autoflex Speed MALDI TOF/TOF mass spectrometer in linearmode using Compas 1.4 control and processing software. The mass spectrawere calibrated by external quadratic calibration using Bruker ProteinCalibration Standard 1 or 2. The sample mass was calculated from theleast charged ion within the calibrated range of the mass spectrum.

Each Alexa-680 molecule added approximately 1000 dalton (˜1 kDa) of massto the activatable antibody. A comparison of the molecular weight of theunconjugated 3954-1204-C225v5 to the conjugated 3954-1204-C225v5 enablesestimation of the number of Alexa-680® molecules that has beenconjugated upon 3954-1204-C225v5.

The MALDI-MW data indicated that up to four 1-kDa molecules wereattached to the activatable anti-EGFR antibody 3954-1204-C225v5. Fromthe reduced samples, it was determined that this modification was mostlikely heterogeneous, as the light chain was observed with 0 and 1modification(s) and the heavy chain was observed with 0, 1 or 2modifications.

It was determined from the MALDI-MS data on digested samples that allpeptides containing cysteine residues were still observed in theconjugated sample (Cyt04-680 high). The MALDI-MW data also confirmedthat the antibody was not fully labeled by Alexa-680®.

Example 4 Materials and Methods

The examples provided herein use an anti-Jagged activatable antibodyreferred to herein as activatable antibody 5342-1204-4D11 (also referredto herein as 5342-1204-4D11 activatable antibody or 5342-1204-4D11) thatincludes a Jagged-binding sequence, a masking moiety (MM), and acleavable moiety (CM) that is a substrate for a protease. It is to beunderstood that while the examples provided herein use these anti-Jaggedactivatable antibody constructs, these methods are applicable to anyactivatable antibody having two or more cysteine residues, where it isdesired that only a portion of the total number of cysteine residues inthe activatable antibody be reduced prior to conjugation. This isreferred to herein as “partial reduction.”

It should be further understood that the examples provided herein use afluorescent agent, Alexa-680 Fluor® (also referred to herein as Alexa680®), as the agent that is to be conjugated to an activatable antibody.This particular dye was chosen because it has a molecular weight that issimilar to a known cytotoxic agent, MMAE. However, this fluorescentagent is merely used as an example, and the compositions and methodsused herein are useful with any number of conjugated agents, includingby way of non-limiting example, toxins and other payload agents. Thecompositions and methods are not limited to agents of any particularmolecular weight, size or other such characteristic.

Anti-Jagged Activatable Antibody Constructs:

The 5342-1204-4D11 activatable anti-Jagged antibody construct includesthe following heavy and light chain sequences:

5342-1204-4D11 Activatable Antibody Heavy Chain Nucleotide Sequence:(SEQ ID NO: 231) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGACCCGGAAGGTCGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATCGGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA5342-1204-4D11 Activatable Antibody Heavy Chain Amino Acid Sequence:(SEQ ID NO: 245) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVIGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSVTRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK5342-1204-4D11 Activatable Antibody Light Chain Nucleotide Sequence:(SEQ ID NO: 233) CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAGGGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 5342-1204-4D11 Activatable Antibody Light ChainAmino Acid Sequence: (SEQ ID NO: 234)QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGLSGRSDNHGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

In some embodiments, the spacer sequence for the light chain5342-1204-4D11 activatable antibody can include an N-terminal variant,such as for example, a spacer selected from the group consisting ofGQSGQ (SEQ ID NO: 235), QSGQ (SEQ ID NO: 236), SGQ (SEQ ID NO: 237), GQand Q. In these embodiments, all other elements of the 5342-1204-4D11activatable antibodies, e.g., the heavy chain sequence, the light chainsequence, the 5342 mask, linker 1, the 1204 substrate, and linker 2, allremain the same as shown above in SEQ ID NO: 234.

Reducing Agent:

The studies provided herein use the reducing agent TCEP(tris(2-carboxyethyl)phosphine).

Protocol for TCEP Partial Reduction of Anti-Jagged Activatable Antibodyand Subsequent Conjugation to Maleimide Alexa-680:

Bond-Breaker@ TCEP Solution (neutral pH solution, Thermo Scientific) isused at various molar ratios of TCEP to an activatable antibody that, inthe cleaved state (i.e., activated state), binds Epidermal Growth FactorReceptor, and the anti-Jagged activatable antibody is formulated in PBS.For example, the ratio of reducing agent, e.g., TCEP, to activatableantibody to be tested can include a ratio in a range from about 20:1 to1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about 9:1to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, fromabout 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5,or from about 1:1 to 1:1.5. In some embodiments, the ratio is in a rangeof from about 5:1 to 1:1. In some embodiments, the ratio is in a rangeof from about 5:1 to 1.5:1. In some embodiments, the ratio is in a rangeof from about 4:1 to 1:1. In some embodiments, the ratio is in a rangefrom about 4:1 to 1.5:1. In some embodiments, the ratio is in a rangefrom about 8:1 to about 1:1. In some embodiments, the ratio is in arange of from about 2.5:1 to 1:1. It is to be understood that while theexamples provided herein use an anti-Jagged activatable antibodyreferred to herein as 5342-1204-4D11, these methods are applicable toany activatable antibody having two or more cysteine residues, where itis desired that only a portion of the total number of cysteine residuesin the activatable antibody be reduced prior to conjugation. This isreferred to herein as “partial reduction.”

Briefly, a TCEP solution at twice the final concentration was mixed 1:1(volume:volume) with 5342-1204-4D11 to result in the finalTCEP:(5342-1204-4D11) ratio desired. The final solution was thenincubated at 37° C. for specified periods of time for the reductionreaction to progress. At the end of the reduction reaction, the solutionwas cooled to room temperature and Maleimide Alexa-680 (Invitrogen) wasadded into the solution (Maleimide Alexa-680 was used at half of thereduction volume and at a concentration equal to 10× molar concentrationof TCEP during the reduction reaction; for example, if the originalreduction reaction comprised 50 microliters (ul) of 13.2 uM5342-1204-4D11 and 50 ul of 52.8 uM TCEP, then 50 ul 264 uM MaleimideAlexa-680 would be used) to begin the Alexa-680 conjugation. Theconjugation reaction proceeded for 2 hours at room temperature in alight tight container. After the 2-hour reaction, the solution was spundown and buffer exchanged into PBS using a PD-10 column (GE Healthcare)or equivalent using manufacturer's instructions. The final conjugatedproduct was analyzed using a UV Spectrophotometer to determine finalprotein concentration and the degree of labeling of the Alexa-680 dye.

Protocol for the Analysis of Maleimide Alexa-680 Conjugated Anti-JaggedActivatable Antibody Using LabChip GXII:

A HT Protein Express LabChip (Perkin Elmer) was prepared according tomanufacturer's instructions using either the Pico Protein Expressprotocol or the HT Protein Express protocol: the Pico Protein Expressprotocol was used to analyze the Alexa-680 conjugated portion of theTCEP reduced 5342-1204-4D11; the HT Protein Express protocol was used toanalyze the total protein in the TCEP reduced, Alexa-680 conjugated5342-1204-4D11. TCEP reduced, Alexa-680 conjugated 5342-1204-4D11 wasprepared for the GXII analysis using Perkin Elmer's instructions. Thesample was analyzed using the 200 series of the LabChip GXII analysisprotocol (High sensitivity for the HT protocol and Pico for the Picoprotocol). Resulting data was analyzed using the LabChip GXII software.

Protocol for Jagged Binding ELISA:

NUNC Maxisorp flatbottom 96 well plates were coated with 50 ul/well, 2ug/ml human Jagged-Fc fusion protein (R&D Systems) in Hank's BalancedSalt Solution (HBSS, Teknova) for 2 hours at room temperature. At theend of the 2 hour coating, the liquid contents of the plate wereevacuated and 250 ul/well of HBSS containing 1% BSA was introduced andallowed to block the plate for 30 minutes at room temperature. At theend of the blocking period, liquid contents of the 96-well plate wereremoved and serially diluted samples (i.e., 5342-1204-4D11, Alexa-680conjugated 5342-1204-4D11, uPA-activated 5342-1204-4D11, uPA-activatedAlexa-680 conjugated 5342-1204-4D11, and 4D111 (an anti-Jaggedantibody), starting at a concentration of 100 ug/ml and diluted by afactor of 3 per dilution step) were introduced at 50 ul/well. The platewas incubated at room temperature for 1 hour. At the end of the hour,the plate was washed with HBSS containing 0.05% Tween-20 using a BioTekELx450 Select CW plate washer (300 ul/well wash volume, 6 cycles ofaspiration and wash). Washed plates were tapped dry and 50 ul/well of400 ng/ml Horse Radish Peroxidase conjugated Goat anti-Human IgG Fab′₂specific antibody (Jackson ImmunoResearch) were introduced and incubatedfor 30 minutes at room temperature. The plates were washed as previouslystated and 100 ul/well of 1-Step TMB Substrate (Thermo Scientific) wasintroduced. Color change was observed and the reaction was stopped bythe addition of 100 ul/well of 1 M HCl (Fisher Scientific). The reactedplate was analyzed using a BioTek EL800 plate reader at O.D. 450. Datawere computed using Excel (Microsoft) and the result was plotted usingPrism 6 (GraphPad).

Example 5 TCEP-Mediated Reduction of Activatable Antibodies

The compositions and methods provided herein determine the combinationof reagents and reaction conditions that produce the desired partialreduction followed by conjugation. When reduction and subsequentconjugation is not controlled properly, activatable antibodies will becompletely reduced, and the masking efficiency of the activatableantibody is compromised.

Studies were conducted to determine the range of reducing agent toactivatable antibody. At lower ratios, for example, in the range of0.5:1 to 2:1 (reducing agent to activatable antibody), some reductionwas achieved, and the activatable antibody integrity and maskingefficiency were retained. At ratios of 1.5:1 to 5:1 (reducing agent toactivatable antibody), reduction time from 30 minutes to 2 hours, therewas an increasing amount of reduced activatable antibody speciescorresponding to the molecular weight of one heavy chain and one lightchain activatable antibody. The partially reduced activatable antibodymaintained the Jagged binding characteristics of the originalnon-reduced and masked activatable antibody demonstrating that theactivatable antibody partially reduced under these conditions wascapable of maintaining the original masking efficiency. At theidentified ratio of reducing agent to activatable antibody and reductiontime, an inter-chain disulfide-reduced activatable antibody can beproduced to allow for subsequent maximum conjugation through freecysteines while maintaining the masking efficiency of the original,non-reduced activatable antibody.

In one set of studies described herein, an activatable anti-Jaggedantibody referred to as 5342-1204-4D11 was reduced at a ratio of TCEP toactivatable antibody equaling 4; 1 using a 120-minute reduction time. Insome instances, reduction was followed by conjugation to a fluorescentdye, Alexa 680. The results of these studies (at TCEP to activatableantibody ratios of 4:1) are shown in FIG. 7.

Using thiol conjugatable Alexa 680 as a surrogate for thiol conjugatabletoxin, these studies demonstrate varying degrees of Alexa 680conjugation dependent on both TCEP to activatable antibody ratio andtime of reduction. The conjugation of thiol conjugatable Alexa 680 toTCEP-partially reduced 5342-1204-4D11 does not significantly change thetitration profile of 5342-1204-4D11 to Jagged. Thus, partial reductionand subsequent thiol conjugation of Alexa 680 can be done in such a wayas to maintain the masking efficiency of activatable antibodies. FIG. 8demonstrates that partial reduction and subsequent thiol conjugation ofAlexa 680 Fluor® can be done in such a way as to also maintainactivation of 5342-1204-4D11 by uPA.

Example 6 Materials and Methods

Antibodies and Activatable Antibodies:

The examples provided herein use an anti-Jagged activatable antibody,referred to herein as activatable antibody 5342-1204-4D11, which isdescribed herein. It is to be understood that while the examplesprovided herein use these anti-Jagged activatable antibody constructs,these methods are applicable to any activatable antibody having two ormore cysteine residues, where it is desired that only a portion of thetotal number of cysteine residues in the activatable antibody be reducedprior to conjugation. This is referred to herein as “partial reduction.”

The examples provided herein also use an anti-Jagged antibody, referredto herein as anti-Jagged antibody 4D11 (also referred to as antibody 411and 411 antibody). The antibody 4D11 includes the following heavy andlight chain sequences:

4D11 Antibody Heavy Chain Nucleotide Sequence: (SEQ ID NO: 231)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGTATTGACCCGGAAGGTCGGCAGACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATCGGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA4D11 Antibody Heavy Chain Amino Acid Sequence: (SEQ ID NO: 245)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK4D11 Antibody Light Chain Nucleotide Sequence:(nucleotides 133-774 of SEQ ID NO: 233)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT4D11 Antibody Light Chain Amino Acid Sequence: (SEQ ID NO: 244)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Additional antibodies used herein include Synagis® (palivizumab) andrituximab, both of which were purchased from Drug Products Service Labs(UCSF) and used as human IgG isotype controls.

SDS Gel:

Five to ten micrograms (ug) of sample were diluted in 7.5 microliters(ul) of water, final volume. 2.5 ul of 4× sample loading buffer(Invitrogen) with or without 1 ul of 10× reducing agent (Invitrogen)were added and the samples heated at ˜90° C. for ˜10 min in a heatingblock. Samples were loaded onto a 10% Bis-Tris (Novex) gel and run inMOPS buffer at 200 mV for ˜40 minutes. Gels were stained in Instant Blue(Expedion) for ˜1 hr followed by de-staining in multiple washes ofwater. Gel images were captured on the Imagequant.

Cell Line:

BxPC3 cells (ATCC CRL 1687) were maintained in CM (RPMI 1640supplemented with 10% heat-inactivated fetal bovine serum (FBS). Priorto FACS binding cell dissociation buffer (Sigma #C5914) was used todissociate the adherent cells.

Protocol for FACS Cell Binding:

The binding of antibodies, activatable antibodies, or theirimmunoconjugate derivatives on BxPC3 cells was evaluated by an indirectimmunofluorescence assay. Dissociated cells (50,000-100,000 per well)were pelleted in a 96-well v-bottom plate and incubated at 4° C. for 45minutes with serial dilutions of test article in 50 to 100 ul of FACSbuffer (FB; HBSS supplemented with 2% FBS). Control wells included humanIgG1 isotype control (Synagis or rituximab) and no IgG containing wells.Cells were washed twice in cold FB and stained with Alexa 647 conjugatedgoat anti-human IgG (AF-647 conjugated affinity pure F(ab′)2 fragmentgoat anti human IgG Fc gamma (γ) fragment specific, Jackson labs,#1909-606-170) for 30 minutes at 4° C. Cells were washed as before,fixed in 1% paraformaldehyde/FB and analyzed using a FACSAria flowcytometer (BD Biosciences). FCS files were analyzed in FCS Express(DeNovo) and mean fluorescence intensity (MFI) against dose titrationplotted in GraphPad PRISM.

Protocol for In Vitro Cytotoxicity Assay:

Cells (4,000 per well) were plated in white walled 96-well plates in 50ul CM. Cells were treated with an equal volume of serial dilutions oftest article for 3 to 5 days. An equal volume (100 ul) of Cell Titer Gloreagent (Promega) was added to each well, according to manufacturer'sinstructions, and relative luminescence units (RLU) was measured on theTecan Infinite M200 Pro. RLU against dose titration was plotted andcurve fitting generated in GraphPad PRISM.

Example 7 Production of Activatable Antibody Conjugates by TCEP-MediatedPartial Reduction and Conjugation

This example describes the use of partial reduction and conjugationmethods of the invention to produce antibody conjugates and activatableantibody conjugates of the invention. It is to be understood that whilethe examples provided herein use anti-Jagged activatable antibodyconstructs, these methods are applicable to any activatable antibodyhaving two or more cysteine residues, where it is desired that only aportion of the total number of cysteine residues in the activatableantibody be reduced prior to conjugation. This is referred to herein as“partial reduction.” In addition, these methods are applicable to anycleavable or non-cleavable linker and agent combination.

This example presents conjugates that display a maleimidePEG-valine-citrulline-para-aminobenzyloxycarbonyl-monomethyl auristatinD linker payload, referred to herein as “vc-MMAD,” or a maleimidePEG-valine-citrulline-para-aminobenzyloxycarbonyl-monomethyl auristatinE linker payload, referred to herein as “vc-MMAE.”

Conjugates of anti-Jagged activatable antibody 5342-1204-4D11 comprisingthe maleimide caproyl-valine-citruline monomethyl auristatin D linkerpayload (vc-MMAD) or the maleimide caproyl-valine-citruline monomethylauristatin E linker payload (vc-MMAE), referred to herein as activatableantibody conjugate 5342-1204-4D11-vc-MMAD and activatable antibodyconjugate 5342-1204-4D11-vc-MMAE, respectively, were prepared asfollows.

The vc-MMAD and vc-MMAE reagents were prepared at Bayside Chemicals(Burlingame, Calif.). N,N, Dimethylacetamide (DMA),tris(2-carboxyethyl)phosphine (TCEP; cat #646547), and IN NaOH werepurchased from Sigma. Prior to performing a conjugation, the followingstock solutions were prepared: 5.0 mg/mL antibody or activatableantibody in PBS (total phosphate=4.25 mM), 5.0 mg/mL vc-MMAD or vc-MMAEin DMA, 1.0 mM TCEP in water, and 10 mM NaOH in water. TCEP was used ata range from 1:1 to 8:1 ratio, typically at a 2.5:1 equimolar ratio, ofTCEP to antibody or activatable antibody.

A typical partial reduction and conjugation was performed as follows,wherein equivalents (eq) are reported relative to the antibody oractivatable antibody. To 200 uL of antibody or activatable antibody inPBS in a tube was added 2.5 eq TCEP. The tube was closed and swirled, togenerate a homogeneous solution, which was allowed to stand for 90minutes at room temperature. The tube was opened, and 13 eq NaOH and 6.0eq vc-MMAD or vc-MMAE were added. The tube was closed and swirled, togenerate a homogeneous solution, which was allowed to stand at roomtemperature for 120 minutes. The tube was opened, and the reactionmixture was passed through a Zeba de-salting column (Thermo Scientific).The protein concentration in the filtrate was analyzed by UVspectrophotometry, and the product was analyzed by SDS gel and HIC-HPLC,and then tested for cell-binding and cell-killing activities. For thecell-binding and cell-killing assays, activatable antibody conjugateswere activated by incubating a 0.5-mg sample of the activatable antibodyconjugate in a 10% solution of recombinant human uPA protease (R and Dsystems MN-207-16) at 37° C. for 16 hr. Protease was removed from thethus activated activatable antibody conjugate by running the samplethrough a pre-washed MabSelect (GE Healthcare) column and eluting with0.1M glycine, followed by neutralization using Tris-HCl. In otherconjugations, the TCEP eq varied from 1.0 to 8.0, and/or the NaOH eqvaried from 0 to 13, and/or the reaction times after the addition ofTCEP, or the additions of NaOH and vc-MMAD or vc-MMAD, varied from 60min to 16 hours.

Example 8 Use of Activatable Antibody Conjugates that IncludeMicrotubule Inhibitor Monomethyl Auristatin D (MMAD)

This example demonstrates that activatable antibody conjugatescomprising microtubule inhibitor MMAD display potent in vitro killingactivity. The example also demonstrates that addition of the linkerpayload to the activatable antibody does not interfere with the abilityof the masking moiety to block binding of the activatable antibodycomprising such masking moiety to its target.

FIG. 9A demonstrates that partial reduction and subsequent thiolconjugation of anti-Jagged activatable antibody 5342-1204-4D11 oranti-Jagged antibody 4D11 with the vc-MMAD linker payload did not affectthe binding behavior of the resultant conjugates to BxPC3 cells: Therewas a 38-fold reduction in IC50 of binding of activatable antibodyconjugate 5342-1204-4D11-vc-MMAD (27 nM) compared to antibody conjugate4D11-vc-MMAD (0.7 nM). Upon activation with uPA, binding of activatedactivatable antibody conjugate 5342-1204-4D11-vc-MMAD was comparable tothat of antibody conjugate 4D11-vc-MMAD. The figure also demonstratesthat Synagis (a human IgG isotype control) did not bind to BxPC3 cells.

Conjugation of activatable antibody 5342-1204-4D11 and antibody 4D11 wasobserved by the increased shift in MW of the HC and LC under reducedconditions by SDS analysis. HIC-HPLC analysis demonstrated between 40 to75% yield of toxin-conjugated material.

FIG. 9B demonstrates the potent killing activity of activatable antibodyconjugate 5342-1204-4D11-vc-MMAD upon activation with uPA and antibodyconjugate 4D11-vc-MMAD. Non-activated activatable antibody conjugate5342-1204-4D11-vc-MMAD killed BxPC3 cells at a 17-fold reduced potency(IC50=7 nM) compared to antibody conjugate 4D11-vc-MMAD (IC50=0.4 nM).The lack of cell killing activity of rituximab antibody (an unrelatedIgG1 isotype antibody) conjugated with the vc-MMAD linker is also shown.Antibody 4D11 alone (i.e., not conjugated) did not exhibit any cytotoxicactivity on BxPC3.

Example 9 Use of Activatable Antibody Conjugates that IncludeMicrotubule Inhibitor Monomethyl Auristatin E (MMAE)

This example demonstrates that activatable antibody conjugatescomprising microtubule inhibitor MMAE display potent in vitro killingactivity. The example also demonstrates that addition of the linkerpayload to the activatable antibody does not interfere with the abilityof the masking moiety to block binding of the activatable antibodycomprising such masking moiety to its target.

FIG. 10A demonstrates that partial reduction and subsequent thiolconjugation of anti-Jagged activatable antibody 5342-1204-4D11 oranti-Jagged antibody 4D11 with the vc-MMAE linker payload did not affectthe binding behavior of the resultant conjugates to BxPC3 cells: Therewas a 33-fold reduction in IC50 of binding of activatable antibodyconjugate 5342-1204-4D11-vc-MMAE (˜30 nM) (which was similar to thebinding activity of non-conjugated activatable antibody 5342-1404-4D11)compared to antibody conjugate 4D11-vc-MMAE (0.9 nM). Upon activationwith uPA, binding of activated activatable antibody conjugate5342-1204-4D11-vc-MMAE was comparable to that of antibody conjugate4D11-vc-MMAE. The figure also demonstrates that Synagis conjugated tovc-MMAE did not bind to BxPC3 cells.

FIG. 10B demonstrates the potent killing activity of activatableantibody conjugate 5342-1204-4D11-vc-MMAE upon activation with uPA andantibody conjugate 4D11-vc-MMAE. Non-activated activatable antibodyconjugate 5342-1204-4D11-vc-MMAE killed BxPC3 cells at a 12-fold reducedpotency (IC50=5 nM) compared to antibody conjugate 4D11-vc-MMAE(IC50=0.4 nM). The lack of cell killing activity of Synagis conjugatedwith the vc-MMAE linker is also shown.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following.

What is claimed is:
 1. A method of partially reducing and conjugating anagent to an activatable antibody resulting in selectivity in theplacement of the agent, the method comprising partially reducing atleast one disulfide bond in the activatable antibody with a reducingagent without disturbing any intrachain disulfide bonds in theactivatable antibody, and conjugating the agent to at least one thiol,wherein the activatable antibody comprises an antibody or an antigenbinding fragment thereof (AB) that specifically binds to a target,wherein the target is Jagged 1 or Jagged 2, a masking moiety (MM) thatinhibits the binding of the AB of the activatable antibody in anuncleaved state to the target, and a cleavable moiety (CM) coupled tothe AB, wherein the CM is a polypeptide that functions as a substratefor a protease.
 2. The method of claim 1, wherein the at least onedisulfide bond is an interchain disulfide bond.
 3. The method of claim1, wherein the at least one disulfide bond is a disulfide bond betweenthe activatable antibody and a second molecule.
 4. The method of claim3, wherein the second molecule is cysteine or glutathione.
 5. The methodof claim 1, wherein the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM or wherein the activatable antibody in theuncleaved state comprises a spacer that is joined directly to the MM andhas the structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.
 6. The method of claim 1, wherein the reducing agent isTCEP.
 7. The method of claim 1, wherein the agent is a toxin or fragmentthereof.
 8. The method of claim 7, wherein the agent is a microtubuleinhibitor or a nucleic acid damaging agent.
 9. The method of claim 7,wherein the agent is a dolastatin or a derivative thereof, an auristatinor a derivative thereof, a maytansinoid or a derivative thereof, aduocarmycin or a derivative thereof, or a calicheamicin or a derivativethereof.
 10. The method of claim 1, wherein the agent is auristatin E ora derivative thereof.
 11. The method of claim 1, wherein the agent ismonomethyl auristatin E (MMAE).
 12. The method of claim 1, wherein theagent is monomethyl auristatin D (MMAD).
 13. The method of claim 1,wherein the agent is DM1 or DM4.
 14. The method of claim 1, wherein theagent is conjugated to the AB via a linker.
 15. The method of claim 14,wherein the linker is a cleavable linker.
 16. The method of claim 1,wherein the agent is a detectable moiety.
 17. The method of claim 16,wherein the detectable moiety is a diagnostic agent.
 18. The method ofclaim 1, wherein the antigen binding fragment thereof is selected fromthe group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, ascAb, a dAb, a single domain heavy chain antibody, and a single domainlight chain antibody.
 19. The method of claim 1, wherein the MM has anequilibrium dissociation constant for binding to the AB that is greaterthan the equilibrium dissociation constant of the AB to the target. 20.The method of claim 1, wherein the MM does not interfere or compete withthe AB for binding to the target when the activatable antibody is in acleaved state.
 21. The method of claim 1, wherein the MM is apolypeptide of no more than 40 amino acids in length.
 22. The method ofclaim 1, wherein the MM polypeptide sequence is different from that ofthe target, and wherein the MM polypeptide sequence is no more than 50/oidentical to any natural binding partner of the AB.
 23. The method ofclaim 1, wherein the CM is a polypeptide of up to 15 amino acids inlength.
 24. The method of claim 1, wherein the protease is co-localizedwith the target in a tissue, and wherein the protease cleaves the CM inthe activatable antibody when the activatable antibody is exposed to theprotease.
 25. The method of claim 1, wherein the activatable antibodycomprises a linking peptide between the MM and the CM, or wherein theactivatable antibody comprises a linking peptide between the CM and theAB.
 26. The method of claim 1, wherein the activatable antibodycomprises a first linking peptide (LP1) and a second linking peptide(LP2), and wherein the activatable antibody in an uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
 27. The method of claim 26,wherein the two linking peptides need not be identical to each other.28. The method of claim 26, wherein each of LP1 and LP2 is a peptide ofabout 1 to 20 amino acids in length.
 29. The method of claim 26, whereinat least one of LP1 or LP2 comprises an amino acid sequence selectedfrom the group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ IDNO: 21), (GGGS)_(n) (SEQ ID NO: 22), GGSG (SEQ ID NO: 23), GGSGG (SEQ IDNO: 24), GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO:27), and GSSSG (SEQ ID NO: 28), where n is an integer of at least one.30. The method of claim 1, wherein the CM is a substrate for an enzymeselected from the group consisting of those shown in Table
 3. 31. Themethod of claim 1, wherein the CM is a substrate for one or more enzymesselected from the group consisting of: a urokinase plasminogen activator(uPA), a legumain, a matriptase, a matrix metalloprotease (MMP), MMP-9,and MMP-14.